Methods and compositions for making and using compatible insecticidal proteins

ABSTRACT

Pesticidal proteins (FFPP&#39;s) are used to produce derivatives (DP&#39;s) that are ineffective and disabled relative to conferring toxic properties upon a target pest, yet the ability of the DP to bind to the receptor to which said FFPP binds is unaffected. Such DP&#39;s are useful in inhibiting the FFPP from which it was derived when both are fed to a target pest and for comparing receptor binding capability and efficiency relative to different FFPP&#39;s from which the DP has been derived, providing for an assessment of different FFPP&#39;s relative to each other, and providing uniformity and certainty in combinations of such FFPP&#39;s for compositions, including transgenic plants, that can be used to control pest populations susceptible to both FFPP&#39;s, creating more durable transgenic plant products, inhibiting the development of resistance to such FFPP&#39;s when used in plants commercially, and in providing a durable and viable resistance management strategy for crops using such FFPP combinations. Polynucleotide sequences intended for use in expression of the DP&#39;s and FFPP&#39;s are also provided. Particular embodiments provide methods of designing and preparing DP&#39;s, as well as compositions and methods of using DP&#39;s and the FFPP&#39;s from which the DP&#39;s have been derived in more effective pesticidal compositions and products.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/640,927, filed Mar. 9, 2018, the entire text of which is specificallyincorporated by reference here in.

INCORPORATION OF SEQUENCE LISTING

A computer readable form of the Sequence Listing is filed herewith byelectronic submission. The Sequence Listing is incorporated by referencein its entirety, is contained in the file created on Mar. 7, 2019 havingthe file name 2019.03.07-38_21_58194 US_0001_ST25.txt, and which is716,181 bytes in size (as measured in the MS-Windows® operating system).

FIELD OF THE INVENTION

The invention generally relates to the field of pest inhibitoryproteins. Generally, a method is provided for selecting two or moretoxin proteins (each being different from the other by at least oneamino acid, and each being toxic to the same target pest) that arecompatible with each other and which can be used collectively (i) in acomposition such as in an agriculturally acceptable formulation fortopical application on, or expressed in, a plant for controlling saidtarget pest; (ii) for diminishing the likelihood of the development ofresistance to any of the compatible toxin proteins; (iii) in apesticidally effective/agriculturally acceptable composition produced inor applied, alone or separately, to a plant; (iv) in a plant or in acomposition for the purposes of protecting a plant from infestation bysaid target pest; (v) in a composition or in a plant for the purpose ofdecreasing the likelihood of the development of resistance by saidtarget pest to any one of the compatible toxin proteins; (vi) in acomposition or in a plant for the purpose of aiding in or improvingresistance management practices for controlling said target pest; (vii)in a composition or in a plant for the purpose of delaying the onset ofresistance to any of the compatible toxins; and in other uniqueapplications for pest management. In particular, the design, productionand useful applications for such compatible protein toxins are provided,as well as compositions and methods for the same.

BACKGROUND OF THE INVENTION

Plant pests, such as numerous species of plant parasitic nematodes,mites, and a plethora of chewing, cutting, boring, and piercing andsucking insects, are the most significant contributors to decreased cropyields. In an attempt to reduce such pest infestation, various chemicaland biological approaches have been developed. Chemical insecticideshave been successful and have been used extensively. However, mostchemical insecticides lack specificity and persist in the environment,exerting toxic effects on non-target species including humans andanimals.

Insect-protected row crops expressing insecticidal proteins (IPs)derived from the entomopathogenic bacterium Bacillus thuringiensis (Bt)have transformed farming practices in many countries (Abrol and Shankar2012). The insecticidal traits resulting from transgene expression ofIPs provide these crops with robust and effective protection from insectherbivory, a benefit that even extends to non-transgenic crops grown inproximity to the transgenic crops, due to area-wide insect pestsuppression (Tabashnik 2010). Bt proteins expressed in plants may beused in their native form or after considerable engineering andimprovement (Siebert 2012, Koch 2015, Badran, Guzov et al. 2016, Gowda2016). Bt protein toxins as well as other toxin proteins that have beenrecently identified from diverse species of microbes, are highlyselective and do not persist in the environment, which is in starkcontrast to chemical approaches for controlling pest infestation,particularly insect pest infestation. Bt toxin proteins, when ingestedby a susceptible insect, become activated by gut proteases, bind tocognate receptors in the insect gut, and form transmembrane pores thateventually kill the insect (Vachon 2012, Pardo-Lopez 2013). A diverseset of such toxin proteins has been discovered. Each protein hasgenerally demonstrated specific toxic activity against a narrow range ofinsect species. For example, Cry1 proteins are observed to exhibit toxiceffects generally against Lepidopteran species, Cry3 proteins aregenerally observed to exhibit toxic effects against Coleopteran species,and yet other more recently identified protein toxins from these sortsof microbes exhibit specific activities against species such asHymenoptera, Diptera, and Heteroptera species. As is the case forsynthetic insecticides, insect pest populations can evolve resistance tocommercially available insecticidal proteins, each of which at one timewere capable of controlling the applicable target pest before thedevelopment of resistance (Tabashnik 2013, Melo 2016). This is largelydue to widespread adoption of crop plants containing such toxin proteinsfor targeted insect pest control, coupled with poor to non-existentresistance management practices, non-compliance with governmentregulatory recommendations, and illegal use activities. There are manymechanisms through which resistance could emerge, but the dominantphenomenon seems to be receptor-mediated wherein the resistant insectsexhibit alterations in key receptors or lower their expression such thatthe toxin is no longer recognized (Tabashnik B. E. 1997, Tabashnik 2013,Melo 2016). Accepted strategies for curtailing insect resistancedevelopment include the planting of a non-transgenic refuge anddeploying insect resistance traits that operate via different mechanismsof action (MOA), which are typically characterized as differences inreceptor binding (Devos, Meihls et al. 2013, Carrière Y. 2015, Deitloff,Dunbar et al. 2016). These methods reduce the chance that a singletarget pest will evolve resistance to one or more of the toxins beingused. The discovery of new efficacious IPs that target insect receptorsdistinct from those that are recognized by currently deployed IPs incommercial insect-protected crops is of paramount importance for thesustainability of this pest management strategy (Zhao J. Z. 2003, BatesS. L. 2005), particularly as a result of increasing human populationsand decreasing availability of arable land.

Theoretically, Bt proteins as well as other toxins derived from microbesthat resemble and/or act in a similar manner as Bt proteins, are allbelieved to initiate their toxic effect upon ingestion by the targetinsect pest of a food source or diet in which the toxic protein ispresent, and thereafter entering the insect gut and binding to brushborder membrane proteins that act as receptors for bringing the toxinclose to the membrane surface. Membrane-bound toxin molecules thenundergo structural transition and likely also, aggregation, to formtransmembrane pores which lead to insect injury (i.e., morbidity) anddeath. Through natural adaptation and selection, pest populations,including insect populations, evolve resistance to such pesticidalproteins. It is believed that alterations in receptor binding by theapplicable toxin is the major mechanism in development of resistant pestspecies, including insect species. As a result, it is important toidentify new IPs for deployment in next-generation insect-protectedcrops that bind to receptors in a target pest, including in a targetinsect pest, that are different in comparison to receptors used by othertoxins which are also effective in the same target pest. (Granero F.1996, González-Cabrera J. 2003, Estela 2004, Jurat-Fuentes 2017). Thereare several published methods used to study IP MOA including ligandblots (Keeton, Francis et al. 1998, Banks, Jurat-Fuentes et al. 2001),in vitro binding experiments with labeled IPs (Jakka 2015) and isolatedinsect gut brush-border membrane vesicle (BBMV) preparations (Martin andWolfersberger 1995), pull-down experiments using immobilized orimmuno-precipitated IPs (Luo, Sangadala et al. 1997), insect cell-basedassays using cloned insect receptor genes (Tanaka 2013, Onofre J. 2017),and the use of resistant insect colonies (Tabashnik B. E. 2000,Tabashnik, Johnson et al. 2000, Herrero, Oppert et al. 2001, Siqueira,Moellenbeck et al. 2004). Apart from resistant colonies, theaforementioned methods provide only a partial representation of an IP'sreceptor preferences due to the highly challenging and complex nature ofthe systems under study. Given the rapidly increasing numbers of knownIPs (Bravo 2012), there is a need for a simple, inexpensive, and robustmethod for MOA differentiation across multiple insect species.

U.S. Pat. No. 5,866,784 (Van Mellaert et al.) discloses combining two ormore Bt's different from each other that bind non-specifically toreceptors on the surface of a target insects' isolated and purifiedbrush border membranes and which do not compete for the same receptor.U.S. Pat. No. 5,500,365 (Fischhoff and Perlak) discloses that Bt toxinsactive against the same insect can be combined to provide at leastadditive insecticidal efficacy, and may provide a synergistic activity,and because of the distinct amino acid sequence between two differentproteins, each may provide a distinct mode of action. The prior artteaches identification of toxins that exhibit toxic effects against asingle target pest using in vitro bioassay methods which includepreparation of brush border membrane vesicles from the specific targetpest, testing a first toxin's ability to bind to the membrane vesiclesand determining a saturation point at which no additional first toxin iscapable of binding the vesicles, then testing a second toxin for itsability to compete with the binding of the first toxin by addingsequentially increasing amounts of said second toxin to a sample ofvesicles saturated with said first toxin, and making a determinationabout whether said second toxin is capable of binding to the samereceptor as said first toxin. There is no mechanism for efficientlyselecting, in vivo in a target pest, two different toxin proteins thateach confer a unique mode of action against the target pest and which donot compete for the same toxin receptor in said pest.

Toxins that each confer morbidity and mortality upon a single targetpest and which are determined by the methods hereunder to bind todifferent receptors in the target pest or which do not interfere withthe steps leading to pesticidal activity are, by default, compatibletoxins, i.e., toxins that belong to a single compatibility group. Twodifferent FFPP's, a first and a second, can be compared forcompatibility by comparing independently the efficacy of any disabledprotein (DP) for reducing lethality (morbidity and or mortality) ofeither FFPP provided in the diet of a target pest and determining thatthe DP interferes with the toxic properties conferred upon a target pestby a first FFPP from which such DP has been derived, and does notinterfere with the toxic properties conferred upon the same target pestby a second FFPP, so long as both FFPP's are independently demonstratedto cause morbidity and mortality to the target pest, such FFPP's arecompatible for use in a composition on or in a plant for controlling thetarget pest by providing at least two different independent modes ofaction.

As a result, the methods disclosed herein and proteins identified asbeing compatible with each other, fulfill a critical unmet need forefficient, effective, rapid discovery and development of effectivecompatible pesticidal proteins for producing pest-free crops that areunlikely to give rise to the development of target pest races which havedeveloped resistance to such toxic proteins.

SUMMARY OF THE INVENTION

The present invention provides for a first polypeptide that exhibitsreceptor binding characteristics that are indistinguishable from that ofa second polypeptide that is different from the first polypeptide by atleast a single amino acid, yet both polypeptides are toxic to the sameinsect species. Except for the receptor binding characteristics of thefirst polypeptide, the first polypeptide inhibits the natural biologicalfunction of the second polypeptide when both are provided together in acomposition. The first polypeptide is devoid of the biological functionof the second polypeptide. The first polypeptide amino acid sequenceexhibits preferably from about 98 percent to about 99.4 percent orgreater identity to the amino acid sequence of the second polypeptideamino acid sequence. The first polypeptide may exhibit a naturalbiological function that is an insecticidal activity against an insectpest of a crop plant. The insect pest may be selected from the groupconsisting of a lepidopteran pest species, a coleopteran pest species, ahemipteran pest species, a homopteran pest species, and a dipteran pestspecies, and the crop plant may be selected from the group consisting ofa dicot and a monocot crop plant. The second polypeptide may be selectedfrom the group consisting of a Bacillus thuringiensis speciesinsecticidal toxin protein, a Brevibacillus species toxin protein, aXenorhabdus species toxin protein, a Photorhabdus species toxin protein,a Bacillus laterosporous species toxin protein, and a Pseudomonasspecies toxin protein.

The present invention also provides a disabled polypeptide (DT) that isderived from a fully functional pesticidal polypeptide (FFPP). Thepesticidal activity of the FFPP on a target pest is dependent upon theFFPP binding to a target pest receptor (TPR) in the gut of the targetpest. The DT is preferably derived by the steps of:

-   -   (a) introducing one or more amino acid sequence modifications        into a pore forming segment of the FFPP to diminish or eliminate        the ability of the FFPP to direct pore formation; optionally the        one or more modifications within the amino acid sequence may be        introduced at a location which results in a reduction of toxic        effect of the resulting DT to the target pest without affecting        binding of the DT to the naturally occurring TPR, and the        resulting modified amino acid sequence of the modified FFPP is        the amino acid sequence of the DT;    -   (b) comparing separately and together the toxic potency of the        unmodified FFPP and the DP when provided alone and in various        proportions together to said target pest;    -   (c) observing that the DP, when used alone, exhibits a        substantially diminished toxic effect upon said target pest        compared to the unmodified FFPP, and    -   (d) observing that the DP, when used in a plurality of molar        ratios with a constant amount of unmodified FFPP, is effective        in titering the toxic effect of the FFPP upon said target pest.

The invention also provides for selecting two or more FFPP's that areeach toxic to a common target insect species, and the two or more FFPP'sthat are selected can be combined together in a composition or combinedtogether for use on or in a plant to control insect infestation of theplant by a target insect pest species. The combination of the pluralityof FFPP's optionally provides (i) for decreasing the likelihood of thedevelopment of resistance by the target insect pest to any of the FFPP'sin the composition; (ii) for aiding in or improving resistancemanagement practices for controlling the target insect pest; or (iii)for delaying the onset of resistance to any of the FFPP's in thecomposition. The two or more FFPP's are preferably selected by the stepsof:

-   -   (a) introducing one or more amino acid sequence modifications        into a pore forming segment of at least one of a first FFPP to        diminish or eliminate said first FFPP's pore formation, (said        one or more modifications being introduced at a location which        results in a reduction of toxic effect of the resulting DP to        the target pest without affecting binding to the naturally        occurring TPR) the resulting modified amino acid sequence        comprising the DT;    -   (b) comparing separately and together the toxic potency of the        unmodified first FFPP and the DP when provided alone and in        various proportions together to said target pest;    -   (c) observing that the DP, when used alone, exhibits a        substantially diminished toxic effect upon said target pest        compared to the unmodified first FFPP;    -   (d) observing that the DP, when used in a plurality of molar        ratios with a constant amount of unmodified first FFPP, is        effective in titering the toxic effect of the first FFPP upon        said target pest; and    -   (e) observing that said DP, when used in a plurality of molar        ratios with a constant amount of an unmodified second FFPP        different from said first FFPP, does not titer the toxic effect        of the second FFPP upon said target pest.        The result is that the first and second FFPP's are thus        compatible for use together in the composition on or in the        plant.

A DP, derived from a FFPP wherein the pesticidal activity of the FFPP onthe target pest depends on the FFPP binding to a target pest receptor(TPR), and wherein the DP has one or more amino acid modifications in adomain of the FFPP at a location which results in reduced toxicity tothe target pest without affecting binding to said TPR when compared tothe FFPP. A method of assessing the mode of action of a first FFPP forcompatibility with a second FFPP to be used in a common pesticidalcomposition, said method comprising the steps of:

-   -   (a) preparing a DP from a first FFPP that is toxic to a target        pest;    -   (b) confirming that said DP when used alone in a bioassay with        said target pest has diminished toxicity against the target pest        when compared to the toxicity of the first FFPP;    -   (c) comparing said DP to a second FFPP different from said first        FFPP alone and in a plurality of molar ratios in which the DP is        present in a greater concentration than said second FFPP in the        diet of the target pest, wherein the inability to titer the        toxic properties of said second FFPP with said DP is        determinative of the binding of said first FFPP and said second        FFPP to different receptors in said target pest.

Also provides are pesticidal protein toxins, wherein the pesticidalactivity of the pesticidal protein toxin is suppressed, partially orfully, in the presence of a polypeptide comprising an amino acidsequence having about 95-99.98% identity to said pesticidal proteinamino acid sequence.

The methods of the present invention provide for a compositioncomprising said first FFPP and said second FFPP is effective incontrolling an insect pest infestation wherein said insects are selectedfrom the group consisting of Arachnida, Coleoptera, Ctenocephalides,Diptera, Hemiptera, Heteroptera, Homoptera, Hymenoptera, Lepidoptera andThysanoptera insects.

The invention also provides for a plant or a seed from a plantcomprising a first recombinant nucleic acid molecule comprising a firstheterologous promoter operably linked to a first polynucleotide segmentencoding a first FFPP and a second recombinant nucleic acid moleculecomprising a second heterologous promoter operably linked to a secondpolynucleotide segment encoding a second FFPP different from said firstFFPP, wherein said first FFPP and said second FFPP are selected for usetogether from the steps as set forth in any of the embodiments above,wherein, optionally:

-   -   (a) said plant or said seed are produced from the breeding        together by the hand of man of two different plants of the same        or substantially similar species, a first plant comprising said        first recombinant nucleic acid molecule expressing said first        FFPP and a second plant comprising said second recombinant        nucleic acid molecule expressing said second FFPP;    -   (b) said plant or seed are produced from the regeneration of a        plant from the transformation of a first plant cell by said        first recombinant nucleic acid molecule and the transformation        of a second plant cell by said second recombinant nucleic acid        molecule;    -   (c) said plant or seed are produced from the transformation of a        single plant cell by said first and said second recombinant        nucleic acid molecule; and    -   (d) said plant or seed are grown from a plant or seed of any of        (a), (b), or (c), wherein said plant or seed comprise said first        and said second recombinant nucleic acid molecule.

The invention provides for compositions comprising a first polypeptideand a second polypeptide that is different from the first polypeptide,wherein said first and second polypeptides are each toxic to a targetpest and do not bind to the same target receptor in said pest, andwherein said first and second polypeptide are selected for use togetherby the steps as set forth in any of the above embodiments.

The invention also provides for methods for, optionally: assessing,selecting, determining the utility of using, determining that: at leasttwo different toxins, pesticidal polypeptides, or pesticidal proteins:are compatible for use together to control a target pest, or, at leasttwo different pesticidal proteins, toxins, or pesticidal polypeptidesare compatible for use together in a single composition, in a plant, orin a mixture, to control a target pest, wherein said method comprises:

-   -   (a) Providing a first pesticidal protein and a second pesticidal        protein different from the first pesticidal protein, wherein        each pesticidal protein is pesticidal when provided alone upon        ingestion to said target pest, and is fully functional in        causing morbidity and/or mortality to said target pest;    -   (b) disabling by modifying/altering/or disrupting the toxic/pore        forming feature of, or inactivating said second pesticidal        protein so that upon ingestion by said target pest, said        disabled second pesticidal protein is unable to cause morbidity        and/or mortality and does not exhibit toxicity to said target        pest (toxins' ability (toxic properties toward said target pest)        to cause any toxic affect upon said target pest is        diminished/significantly diminished or reduced/eliminated)        without (diminishing/reducing/affecting/eliminating) said        disabled second toxins' capacity to bind (ability to        bind/affinity for binding) a particular receptor in the gut of        said target pest to which said second toxin normally binds (said        second toxin normally has affinity);    -   (c) providing in the diet of said target pest a sufficient        amount of said disabled second toxin to bind        (mask/block/reduce/eliminate) the particular receptor in the gut        of said target pest to which said second toxin (said disabled        second toxin) normally binds (has affinity to);    -   (d) providing in the diet of said target pest to which said        sufficient amount of said disabled second toxin has been        provided (of step (c)), an amount (a pesticidally effective        amount) of said first toxin (that is known from step (a))        sufficient to elicit a toxic effect upon said target pest; and    -   (e) Observing the effects of said first toxin upon said target        pest in the presence of said disabled second toxin;    -   wherein an observation from step (e) that said first toxin        exhibits a toxic effect is determinative that said first toxin        and said second toxin are compatible for use together.

The invention also provides for a method for assessing the contributionof a toxin protein to the overall efficacy of a composition containingtwo or more toxins which are different from each other and are eachtoxic to the same target insect pest, comprising the steps of:

-   -   (a) Providing a first toxin and a second toxin different from        the first, wherein each toxin is toxic upon ingestion (in the        absence of the other toxin) to said target pest (is fully        functional in causing morbidity and/or mortality to said target        pest);    -   (b) disabling (modifying/altering/disrupting the toxin feature        of/inactivating) said second toxin so that upon ingestion by        said target pest, said disabled        (altered/modified/disrupted/inactivated) second toxin fails to        cause morbidity and/or mortality (does not exhibit toxicity to        said target pest/toxins' ability (toxic properties toward said        target pest) to cause any toxic affect upon said target pest is        diminished/significantly diminished or reduced/eliminated)        without diminishing (reducing/affecting/eliminating) said        disabled second toxins' capacity to bind (ability to        bind/affinity for binding) a particular receptor in the gut of        said target pest to which said second toxin normally binds (said        second toxin normally has affinity);    -   (c) providing in the diet of said target pest a sufficient        amount of disabled second toxin to bind        (mask/block/reduce/eliminate) the particular receptor in the gut        of said target pest to which said second toxin (said disabled        second toxin) normally binds (have affinity to);    -   (d) providing in the diet of said target pest to which said        sufficient amount of said disabled second toxin has been        provided (of step (c)), an amount (a pesticidally effective        amount) of said first toxin (that is known from step (a))        sufficient to elicit a toxic effect upon said target pest; and    -   (e) Observing the effects of said first toxin upon said target        pest in the presence of said disabled second toxin; wherein an        observation from step (e) that said first toxin exhibits a toxic        effect is determinative that said first toxin and said second        toxin are compatible for use together in a pesticidal        composition, expressed in, or applied to a plant for:        -   (1) Controlling said target pest; (2) Protecting said plant            from infestation by said target pest; or        -   (3) decreasing the likelihood of the development of            resistance by said target pest to either the first toxin or            the second toxin        -   (4) for the purpose of aiding in or improving resistance            management practices for controlling said particular target            pest; or        -   (5) in a composition or in a plant for the purpose of            delaying the onset of resistance to any of the compatible            toxins.

Any of the methods above are contemplated to provide for a first toxinor pestidical protein having a naturally occurring receptor bindingmotif and a second toxin or pesticidal protein is a naturally occurringprotein or is an engineered insecticidal protein (chimera, modified(insertion, deletion, substitution of one or more amino acids))engineered using any number of methods including (i) site directedmodification of a gene encoding such protein to cause the insertion,deletion or substitution of one or more amino acids, (ii) directedevolution methods of Maxygen, Verdia, or those in which phage, inparticular filamentous bacteriophage, are used, and (iii) randommutagenesis and selection of a functional toxin protein.

The invention also provides for a plant comprising a combination of twoor more different toxin/pesticidal proteins each toxic to the sametarget pest, wherein said toxin/pesticidal proteins have been selectedfor use in such plant using steps as set forth in any of the precedingembodiments, and for a composition for use in controlling a target pest,wherein said composition comprises at least two differenttoxin/pesticidal proteins selected for use in such composition using thesteps as set forth in any of the preceding embodiments.

The invention provides for seed of a plant, wherein the genome of saidseed comprises a first transgene encoding a first toxin/pesticidalprotein and a second transgene encoding a second toxin/pesticidalprotein, wherein either toxin/pesticidal protein alone is effective incontrolling the same target pest, wherein the two toxins/pesticidalproteins have been selected for use together in the same plant using anyof the steps as set forth in any of the preceding embodiments, and theplants that are contemplated are further selected from the groupconsisting of a monocot and a dicot; wherein said monocot is furtherselected from the group consisting of corn, wheat, rice, and millets,and wherein said dicot is further selected from the group consisting ofsoybean, cotton, sunflower, alfalfa, canola, pigeon pea, tomato, pepper,gourd, melon, apple, pear, fig, orange, grapefruit, lemon, lime, andperennial flowers.

The invention provides for a method for selecting two or more toxinproteins (each being different from the other by at least one aminoacid, and each being toxic to the same target pest) that are compatiblewith each other and which can be used collectively and optionally:

-   -   (a) in a composition or in a plant for controlling said target        pest;    -   (b) for diminishing the likelihood of the development of        resistance to any of the compatible toxin proteins;    -   (c) in a pesticidally effective/agriculturally acceptable        composition produced in or applied, alone or separately, to a        plant;    -   (d) in a plant or in a composition for the purposes of        protecting a plant from infestation by said target pest;    -   (e) in a composition or in a plant for the purpose of decreasing        the likelihood of the development of resistance by said target        pest to any of the compatible toxin proteins;    -   (f) in a composition or in a plant for the purpose of aiding in        or improving resistance management practices for controlling        said target pest;    -   (g) in a composition or in a plant for the purpose of delaying        the onset of resistance to any of the compatible toxins; and    -   (h) in other unique applications for pest management.

In particular, the design, production and useful applications for suchcompatible protein toxins are provided, as well as compositions andmethods for the same. The method also provides for selecting toxinstoxic to the same pest for use together in a composition or in plantsfor protecting against the infestation of the pest, and to reduce thelikelihood of development of resistance of the pest to either of theselected toxins used together in the composition or in the plant.

A method of preparing a disabled polypeptide (DP) from a first fullyfunctional pesticidal polypeptide (first FFPP) is provided in which thepesticidal activity of the first FFPP on the target pest depends on theFFPP binding to a naturally occurring target pest receptor (TPR). Themethod comprises the steps of:

-   -   (a) First, introducing one or more modifications into a segment        of the first FFPP to produce a DP; the one or more modifications        are introduced at a location in the amino acid sequence of the        first FFPP which results in the formation of a new amino acid        segment sequence, the sequence of the resulting DP. The DP        exhibits a toxic potency substantially less than the toxic        potency of the first FFPP when the DP is provided separately in        the diet of the target pest. The ability of the DP to bind to        the naturally occurring TPR to which the first FFPP binds is        unaffected; and    -   (b) Second, the first FFPP and the DP are then compared        separately to confirm the reduction or absence of toxic potency        of the DP to the target pest, and to evaluate the ability or        extent to which the DP is able to reduce or eliminate the toxic        potency of the first FFPP by first providing the DP in the diet        of a plurality of the target pest and then provide samples        containing the first FFPP at various concentrations or        proportions to separate groups of the plurality of pests to        which the DP has been provided;        And observing whether the DP, when used alone, exhibits a        substantially diminished toxic potency compared to that of the        first FFPP when provided in the diet of said target pest, and        observing whether and the extent to which the DP, when used        together with various molar ratios/concentrations of said first        FFPP, is effective in titering the toxic potency of the first        FFPP toward the target pest.

Disabled insecticidal protein toxin isoforms, capable of binding theappropriate receptor but incapable of inducing or conferring toxiceffects, are derived from insecticidal protein toxins (parent toxins,fully functional isoform toxins), such that the disabled toxins retainthe parent toxins ability to bind to a target insect receptor proteinbut are incapable of undergoing all further changes required to kill orstunt the target insect, i.e., incapable of inducing or conferring toxiceffects. For example, Bt derived insect toxins, following ingestion,activation and binding to its cognate or natural receptor in the targetinsect gut, undergo structural changes to form oligomers which insertinto the insect membrane, forming transmembrane pores that result infeeding cessation and death. The disabled insecticidal protein toxin'sability to compete with the parent toxin, or fully functional isoformtoxin, at the insect protein binding site (the cognate or normal,natural receptor), without causing, conferring or conveying toxiceffects (i.e., inhibiting stunting or mortality), provides a useful toolfor discovering toxins which bind to different insect toxin receptorproteins and thereby exhibit a different mode of action, compared to thefully functional isoform toxin. Combining insect toxins which displaydifferent modes of action by binding to different toxin receptorproteins in the same insect species has proven useful in developingcrops that survive insect infestation without allowing or encouragingtoxin-resistance in the target insect species.

Methods for preparing disabled insecticidal proteins are providedherein. In one embodiment the disabled insecticidal protein toxin isprepared by introducing one or more changes in amino acids at one ormore positions within the amino acid sequence of the fully functionalparent or isoform toxin, at a location which results in reduced toxicityto the target insect without effecting disabled protein binding to thenatural protein receptor in the target insect gut. In the case of athree-domain Cry toxin, the amino acid changes are in Domain I, which isbelieved to be the site of transmembrane pore formation. Domains II andIII are not changed so that the insect receptor protein bindingproperties are retained in the disabled protein toxin. In anotherembodiment the disabled insecticidal protein toxin is derived from aβ-pore-forming Bt insecticidal protein toxin. In this case, toxic poreformation is prevented by introducing changes in amino acids positionedin the amphipathic β-pore-forming loop or in an adjoining proteinstructure or in both.

In additional embodiments the disabled insecticidal protein toxin isprepared by introducing at least two cysteine mutations into regions ofthe insecticidal protein toxin involved in toxic pore formation, such asthose described above. The cysteine mutations can be introduced aseither replacement mutations, substituting for amino acids in thesequence of the fully functional toxin, or insertion mutations, addingto the sequence at positions between two existing amino acids, with theproviso that these at least two cysteine residues are located 8 to 10angstroms apart. Subsequently reacting the mutated protein with abifunctional sulfhydryl crosslinking reagent, such as iodoacetamidederivative N,N′-ethylene bis (iodoacetamide), maleimide derivative bis(maleimido) ethane or the like, results in a derivative of the fullyfunctional toxin which retains the ability to bind to the natural ornormal target toxin receptor without producing a toxic effect on thetarget insect.

In another embodiment the disabled proteins are screened for activity ina diet bioassay against the target insect. First, it is established thatthe disabled protein demonstrates a lack of toxicity in the targetinsect by comparing results from two separate diet bioassays, one usingthe disabled protein toxin isoform, and the other using the fullyfunctional isoform toxin, both at equivalent concentrations that producea toxic effect when using the fully functional toxin, and no toxiceffect when using the disabled protein isoform. Second, the ability ofthe disabled protein toxin to retain toxin receptor protein binding isdemonstrated in a diet bioassay against the target insect.Administration of various molar ratios of the disabled protein and thefully functional isoform toxin are mixed together in the insect diet.Results exhibit a decrease in target insect toxicity for the fullyfunctional protein toxin where the molar concentration of the disabledprotein is equal to or greater than the molar concentration of the fullyfunctional toxin.

Isolated disabled protein toxins are provided, derived from fullyfunctional isoform toxins, with one or more amino acid modifications,including substitutions, insertions and deletions, in a protein domainof the fully functional isoform toxin, at a location which results inreduced toxicity to the target insect without effecting target insectreceptor protein binding when compared to the original, fully functionalisoform toxin. Disabled insecticidal protein toxins disclosed hereininclude those set forth in SEQ ID NOs:4 and 6, encoded by nucleotidesequences set forth in SEQ ID NOs:3 and 5, respectively.

In another embodiment, isolated disabled protein toxins are provided,derived from fully functional isoform protein toxins, with at least twocysteine amino acid substitutions located 8 to 10 angstroms apart in aprotein domain of the fully functional isoform toxin, which is rendereddisabled following exposure to a bifunctional sulfhydryl crosslinkingreagent, such as iodoacetamide derivative N,N′-ethylene bis(iodoacetamide), maleimide derivative bis (maleimido) ethane or thelike, resulting in a derived variant of the fully functional isoformtoxin which retains the ability to bind to the normal target insecttoxin receptor without causing any toxic effect on the target insect.Disabled insecticidal protein toxins disclosed herein include those setforth in SEQ ID NOs:8, 12, 16, 22, 24, 26 and 28, encoded by nucleotidesequences set forth in SEQ ID NOs:7, 11, 15, 21, 23, 25 and 27,respectively.

Also provided herein is a method of assessing the mode of action for anyparticular insecticidal protein toxin against a target insect bycomparing the modes of action of a plurality of different insecticidalprotein toxins in the target insect, wherein the mode of action isdistinguished by the binding of an individual fully functionalinsecticidal protein toxin to a specific target insect receptor protein.Thus, toxins shown to bind to the same insect receptor protein share thesame mode action, while toxins that bind to different insect receptorproteins display different modes of action. The assessment is carriedout as follows: (a) prepare a disabled protein toxin, as describedabove, for a particular fully functional protein toxin; (b) evaluate thetoxicity of the particular fully functional protein toxin against thetarget insect, in the absence and in the presence of a molar excess ofthe disabled protein toxin isoform; and (c) compare the resulting targetinsect susceptibility to protein toxin effects to assess the mode ofaction for the particular protein toxin, wherein the mode of action forthe protein toxin is the same as the fully functional isoform toxin ofany disabled protein toxin which suppresses or impairs the toxicactivity of the particular protein toxin. Disabled insecticidal proteintoxins disclosed herein include those set forth in SEQ ID NOs: 4, 6, 8,12, 16, 22, 24, 26 and 28.

In another embodiment, fully functional protein toxins are providedwhose toxicities are partially or fully suppressed or impaired in thepresence of a polypeptide comprising an amino acid sequence having about44%-100%, including 44%, or 50%, or 55%, or 60%, or 65%, or 70%, or 75%,or 80%, or 85%, or 90%, or 95%, or 99%, or 100%, amino acid sequenceidentity to the amino acid sequence of any of SEQ ID NOs:4, 6, 8, 12,16, 22, 24, 26 and 28. In another embodiment these insecticidal proteintoxins, identified as described above, provide a method for controllinginsect pest infestation by contacting the insect pest with an insectinhibitory amount of these insecticidal protein toxins, which areespecially useful in controlling pest infestations by Coleoptera,Diptera, Hymenoptera, Hemiptera and Lepidoptera. In another embodimentthese insecticidal protein toxins, identified as described above, can beexpressed in a plant or a seed from a plant, providing protection frominsect infestation by incorporating a recombinant nucleic acid moleculecomprising a heterologous promoter operably linked to a polynucleotidesegment encoding one of these identified insecticidal protein toxins.

In another embodiment is a method for selecting two pesticidal agentscompatible for use together in a composition for controlling a targetpest, said method comprising the first step of selecting a first and asecond toxic agent, each agent being different from the other and eachagent causing toxic properties when provided individually in the diet ofa target pest. A second step includes producing a DT from said firsttoxic agent that, upon ingestion by said target pest, blocks the toxicproperties conferred by said first toxic agent but does not itselfconfer toxic properties. The third step in the method provides forproducing a plurality of different mixtures containing a fixed butpesticidally effective amount of a second toxic agent, and increasingamounts of said DT. The next step provides in the diet of said targetpest, a pesticidally effective amount of said second toxic agent, thenproviding a dose of each mixture of the third step separately to each ofat least three different individuals of said target pest. The targetpests having received the various samples or doses of toxic agent or oftoxic agent mixtures is then observed for evidence of any toxicproperties in any of the individual target pests that have received thevarious doses, and any such observation of such toxic properties isdeterminative that said first and second toxic agents are compatible foruse together to control the said target pest.

The method can further include a recombinant plant or seed expressingtwo or more pesticidal agents, wherein the said agents are selected foruse together as compatible agents according to the method described inparagraph [0033] above.

The invention includes embodiments such as a method for selecting afirst FFPP and a second FFPP to be combined together in a composition orfor use on or in a plant to control insect infestation of said plant bya target insect pest species, wherein said combination of said FFPP'soptionally provide:

-   -   (a) for decreasing the likelihood of the development of        resistance by said target insect pest to any of the FFPP's in        said composition;    -   (b) for aiding in or improving resistance management practices        for controlling said target insect pest; or    -   (c) for delaying the onset of resistance to any of the FFPP's in        said composition.

The method provides for the said two or more FFPP's to be selected foruse together by the steps as set forth in paragraph [0033] above, andthe DP, when used in a plurality of molar ratios with a constant amountof said first FFPP, is effective in titering the toxic effect of thefirst FFPP upon said target pest. The said DP, when used in a pluralityof molar ratios with a constant amount of said second FFPP, does nottiter the toxic effect of the second FFPP upon said target pest. In suchcase, the said first and said second FFPP are compatible for usetogether in said composition on or in said plant.

Another method of the invention provides for assessing the mode ofaction of a first FFPP for compatibility with a second FFPP to be usedin a common pesticidal composition, said method comprising the steps offirst preparing a DP from a first FFPP that is toxic to a target pest,then confirming that said DP when used alone in a bioassay with saidtarget pest has diminished toxicity against the target pest whencompared to the toxicity of the first FFPP, and finally comparing saidDP to a second FFPP different from said first FFPP, the second FFPPalone or the first FFPP alone but in each case along with a variableamount of said DP, i.e., for each FFPP/DP combination, in a plurality ofmolar ratios in which the DP is present in a greater concentration thansaid second FFPP in the diet of the target pest. Observing in the laststep the inability of the DP present in any amount in a combination withthe second FFPP to titer (to reduce, to inhibit, or to suppress) thetoxic properties of said second FFPP with said DP is determinative ofthe binding of said first FFPP and said second FFPP to differentreceptors in said target pest, and therefore a functional assessment ofthe mode of action of a first FFPP for compatibility with a second FFPPto be used in a common pesticidal composition.

Additionally, the methods set forth in paragraphs [0035] and [0037] maybe further defined as a composition comprising said first FFPP and saidsecond FFPP, the composition being effective in controlling an insectpest infestation wherein said insects are selected from the groupconsisting of Arachnida, Coleoptera, Ctenocephalides, Diptera,Hemiptera, Heteroptera, Homoptera, Hymenoptera, Lepidoptera andThysanoptera insects. The method set forth in paragraph [0033] may befurther defined as a composition comprising said first toxic agent andsaid second toxic agent, the composition being effective in controllingan insect pest infestation wherein said insects are selected from thegroup consisting of Arachnida, Coleoptera, Ctenocephalides, Diptera,Hemiptera, Heteroptera, Homoptera, Hymenoptera, Lepidoptera andThysanoptera insects.

The invention further contemplates a plant or a seed from a plantcomprising a first recombinant nucleic acid molecule comprising a firstheterologous promoter operably linked to a first polynucleotide segmentencoding a first FFPP and a second recombinant nucleic acid moleculecomprising a second heterologous promoter operably linked to a secondpolynucleotide segment encoding a second FFPP different from said firstFFPP, wherein said first FFPP and said second FFPP are selected for usetogether from the steps of any of the foregoing methods. Optionally, thesaid plant or said seed are produced from the breeding together by thehand of man of two different plants of the same or substantially similarspecies. A first plant may comprise said first recombinant nucleic acidmolecule expressing said first FFPP and a second plant may comprise saidsecond recombinant nucleic acid molecule expressing said second FFPP.The said plant or seed are produced from the regeneration of a plantfrom the transformation of a first plant cell by said first recombinantnucleic acid molecule and the transformation of a second plant cell bysaid second recombinant nucleic acid molecule (or by transformation of asingle cell by both recombinant molecules, whether operably linkedtogether or whether each molecule is separate from the other), and thesaid plant or seed are grown from a plant or seed of any of the stepsabove in this paragraph, wherein said plant or seed comprise said firstand said second recombinant nucleic acid molecule.

Compositions are contemplated which may comprise a first polypeptide anda second polypeptide that are each different from each other, and saidfirst and second polypeptides are each toxic to a target pest and do notbind to the same target receptor in said pest, and wherein said firstand second polypeptide are selected for use together by the steps of,first, producing a DT from said first polypeptide that, upon ingestionby said target pest, blocks the toxic properties conferred by said firstpolypeptide but does not itself confer toxic properties; second,producing a plurality of different mixtures containing a fixed butpesticidally effective amount of said second polypeptide, and increasingamounts of said DT; third, providing in the diet of said target pest, apesticidally effective amount of said second polypeptide; fourth,providing a dose of each mixture of the third step separately to each ofat least three different individuals of said target pest; and then last,observing toxic properties in any individual in the fourth step. Anobservation of toxic properties of the second polypeptide in such pestwould be determinative that said first and second polypeptides arecompatible for use together to control said target pest.

Other embodiments, features, and advantages of the invention will beapparent from the following detailed description, the examples and theclaims.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 is a nucleotide sequence encoding a Cry1Ab3 protein.

SEQ ID NO:2 is an amino acid sequence of a Cry1Ab3 protein.

SEQ ID NO:3 is a nucleotide sequence encoding a disabled Cry1Ab protein.

SEQ ID NO:4 is an amino acid sequence of a disabled variant Cry1Ab3_2protein.

SEQ ID NO:5 is a nucleotide sequence encoding a disabled variantCry1Ab3_3 protein.

SEQ ID NO:6 is an amino acid sequence of a disabled variant Cry1Ab3_3protein.

SEQ ID NO:7 is a nucleotide sequence encoding a disabled variantCry1Ab3_4 protein.

SEQ ID NO:8 is an amino acid sequence of a disabled variant Cry1Ab3_4protein.

SEQ ID NO:9 is a nucleotide sequence encoding a TIC105 protein.

SEQ ID NO: 10 is an amino acid sequence of a TIC105 protein.

SEQ ID NO: 11 is a nucleotide sequence encoding a disabled variantTIC1053 protein.

SEQ ID NO: 12 is an amino acid sequence of a disabled variant TIC1053protein.

SEQ ID NO: 13 is a nucleotide sequence encoding a TIC107 protein.

SEQ ID NO: 14 is an amino acid sequence of a TIC107 protein.

SEQ ID NO: 15 is a nucleotide sequence encoding a disabled variantTIC107_4 protein.

SEQ ID NO: 16 is an amino acid sequence of a disabled variant TIC107_4protein.

SEQ ID NO: 17 is a nucleotide sequence encoding a Cry2Ab2 protein.

SEQ ID NO: 18 is an amino acid sequence of a Cry2Ab2 protein.

SEQ ID NO: 19 is a nucleotide sequence encoding a TIC834_16 protein.

SEQ ID NO:20 is an amino acid sequence of a TIC834_16 protein.

SEQ ID NO:21 is a nucleotide sequence encoding a disabled variantTIC834_18-1 protein.

SEQ ID NO:22 is an amino acid sequence of a disabled variant TIC834_18-1protein.

SEQ ID NO:23 is a nucleotide sequence encoding a disabled variantTIC834_21-1 protein.

SEQ ID NO:24 is an amino acid sequence of a disabled variant TIC834_21-1protein.

SEQ ID NO:25 is a nucleotide sequence encoding a disabled variantTIC834_22-1 protein.

SEQ ID NO:26 is an amino acid sequence of a disabled variant TIC834_22-1protein.

SEQ ID NO:27 is a nucleotide sequence encoding a disabled variantTIC834_23-1 protein.

SEQ ID NO:28 is an amino acid sequence of a disabled variant TIC834_23-1protein.

SEQ ID NO:29 is a nucleotide sequence encoding a disabled variantCry2Ab2_6 protein.

SEQ ID NO:30 is an amino acid sequence of a disabled variant Cry2Ab2_6protein.

SEQ ID NO:31 is a nucleotide sequence encoding a TIC834_14 ßFFPP-2protein.

SEQ ID NO:32 is an amino acid sequence of a TIC834_14 ßFFPP-2 protein.

SEQ ID NO:33 is an artificial nucleotide sequence encoding a BCW003toxin protein.

SEQ ID NO:34 is an amino acid sequence of a BCW003 toxin protein.

SEQ ID NO:35 is an artificial nucleotide sequence encoding a disabledvariant BCW003 referred to as DT11.

SEQ ID NO:36 is an amino acid sequence of a disabled variant BCW003protein DT11.

SEQ ID NO:37 is a nucleotide sequence encoding a Cry1Ca toxin protein.

SEQ ID NO:38 is an amino acid sequence of a Cry1Ca toxin protein.

SEQ ID NO:39 is a nucleotide sequence encoding a disabled variant ofCry1Ca, referred to as DT12.

SEQ ID NO:40 is an amino acid sequence of a disabled toxin Cry1Careferred to as DT12.

SEQ ID NO:41 is a nucleotide sequence encoding a TIC844 toxin protein.

SEQ ID NO:42 is an amino acid sequence of a TIC844 toxin protein.

SEQ ID NO:43 is a nucleotide sequence encoding a disabled TIC844 toxinprotein referred to as DT13.

SEQ ID NO:44 is an amino acid sequence of a disabled toxin TIC844referred to as DT13.

SEQ ID NO:45 is a nucleotide sequence encoding a TIC868 toxin protein.

SEQ ID NO:46 is an amino acid sequence of a TIC868 toxin protein.

SEQ ID NO:47 is a nucleotide sequence of a disabled TIC868 toxin proteinreferred to as DT14.

SEQ ID NO:48 is an amino acid sequence of a disabled TIC842 proteinreferred to as DT14.

SEQ ID NO:49 is a nucleotide sequence encoding a TIC842 toxin protein.

SEQ ID NO:50 is an amino acid sequence of a TIC842 toxin protein.

SEQ ID NO:51 is a nucleotide sequence of a disabled TIC842 toxin proteinreferred to as DT15.

SEQ ID NO:52 is an amino acid sequence of a disabled TIC868 proteinreferred to as DT15.

SEQ ID NO:53 is a nucleotide sequence encoding a VIP3A toxin protein.

SEQ ID NO:54 is an amino acid sequence of a VIP3A toxin protein.

SEQ ID NO:55 is a nucleotide sequence of a disabled VIP3A toxin proteinreferred to as DT16.

SEQ ID NO:56 is an amino acid sequence of a disabled VIP3A proteinreferred to as DT16.

SEQ ID NO:57 is a nucleotide sequence encoding a TIC1100 toxin protein.

SEQ ID NO:58 is an amino acid sequence of a TIC1100 toxin protein.

SEQ ID NO:59 is a nucleotide sequence of a disabled TIC1100 toxinprotein referred to as DT17.

SEQ ID NO:60 is an amino acid sequence of a disabled TIC1100 referred toas DT17.

SEQ ID NO:61 is a nucleotide sequence encoding a TIC867 toxin protein.

SEQ ID NO:62 is an amino acid sequence of a TIC867 toxin protein.

SEQ ID NO:5635 is a nucleotide sequence of a disabled TIC867 toxinprotein referred to as DT18.

SEQ ID NO:64 is an amino acid sequence of a disabled TIC867 referred toas DT18.

DETAILED DESCRIPTION OF THE INVENTION

One of the most challenging problems facing researchers in the field ofinsecticidal toxin discovery and development involves the identificationof new modes of action (MOA) for insect resistance management.Alterations in receptor binding sites are believed to play a significantrole in the development of toxin resistance by insects in the field.Methods that distinguish differences in receptor binding include ligandblotting and competitive binding assays, using isolated insect brushborder membranes (BBMs). These methods require significant developmentwork to optimize both toxin- and BBM-preparation and to validate theseassays across multiple insect species. The methods described in thisdisclosure rely on the use of disabled insecticidal proteins or disabledtoxins that retain insect receptor binding activity but are unable toproduce a toxic or lethal effect in the target insect. Such disabledinsecticidal proteins are able to compete with homologous native toxinsin insect bioassays, resulting in suppression of insecticidal activityby reducing or preventing the native or parent toxin from binding to acognate insect receptor, required to produce a lethal effect on thetarget insect species. When combined with heterologous insect toxinsthat don't share a common MOA, such as by not binding to the samereceptor as the disabled insecticidal protein, these disabledinsecticidal proteins should not exhibit competitive inhibition ininsect bioassays. The outcome of these competitive assays is unambiguousand requires only a functional insect bioassay including the cognateinsect receptor which binds the parent toxin. This method thus providesa facile procedure for researchers to arrange Cry proteins or otherinsecticidal toxins into groups that are likely to share receptorbinding sites. While this data alone doesn't provide conclusive evidencethat two toxins share receptor binding sites, it does allow researchersto prioritize those toxins that appear to operate through differentmodes-of-action based upon the absence of competition in an insectbioassay that includes a disabled insecticidal protein. In addition,since the method only requires a valid insect bioassay, it is easier toassess competition across a wide range of insect target species.

Unless otherwise noted, terms are to be understood according toconventional usage by those of ordinary skill in the relevant art.

As used herein a “disabled pesticidal protein” or “disabled toxin” is aprotein, derived from an insect toxin, such as a Bt protein toxin, whichretains the capability of insect receptor binding without producing alethal effect on the target insect.

The terms “active” or “activity”; “pesticidal activity” or “pesticidal”;“entomocide” or “entomocidal”; “nematicide” or “nematicidal”;“fungicide” or fungicidal”; “insecticidal activity”, “insectinhibitory”, “insecticidal”, or “an insect inhibitory amount”, refer toefficacy of a toxic agent, such as an insecticidal protein, ininhibiting (inhibiting growth, feeding, fecundity, or viability),suppressing (suppressing growth, feeding, fecundity, or viability),controlling (controlling the pest infestation, controlling the pestfeeding activities on a particular crop containing an effective amountof a disclosed insecticidal or pesticidal protein) or killing (causingthe morbidity, mortality, or reduced fecundity of) an applicable targetpest.

Reference to a pest, particularly a pest of a crop plant, means, forexample, nematode, fungal or insect pests and the like of crop plants,particularly any embryonic, larval, nymph or adult form of an Arachnid,Coleopteran, Ctenocephalides, Dipteran, Hemipteran, Heteropteran,Homopteran, Hymenopteran, Lepidopteran or Thysanopteran insect. The term“target pest” refers to a particular pest species for which a pesticidalprotein is selectively toxic.

As used herein, a “transgenic plant”, “transgenic plant event” or“transgenic crop” is any plant in which one or more, of the cells of theplant include a transgene. A transgene may be integrated within anuclear genome or organelle genome, or it may be extra-chromosomallyreplicating DNA. The term “transgene” means a nucleic acid that ispartly or entirely heterologous or foreign to a plant or cell into whichit is introduced.

Reference to “resistance”, as in “insect resistance” or “pestresistance”, refers to the development of one or more mechanisms in aninsect, nematode or fungal pest to overcome or nullify the lethaleffects of a pest toxin. For protein toxins, such as Bt toxins, that mayrequire binding to an insect receptor protein to initiate a lethalevent, resistance may develop when there is a change in the toxins aminoacid sequence, for example as caused by a mutation, such as an aminoacid substitution, insertion or deletion in the target insect toxinreceptor protein, preventing or inhibiting the toxin from binding to thetarget insect receptor protein.

The term “parent toxin”, “native toxin”, “wild-type toxin”, “fullyfunctional toxin” or “fully functional isoform toxin” refers to thepesticidal protein toxin from which a disabled pesticidal protein isderived. Design and production of the disabled insecticidal orpesticidal protein depends on the particular structure of the parent orfully functional isoform toxin using methods, including but not limitedto introducing changes in the amino acid sequence, through insertion,deletion or substitution at one or more positions in the sequence of thefully functional isoform toxin, in order to render the disabled toxinnon-lethal, while retaining the capability to bind to the natural pestreceptor protein to which the fully functional toxin binds to initiatelethal activity. In addition, chemical crosslinks may be introduced atsusceptible sites in the toxin sequence, such as bifunctional sulfhydrylcrosslinks between two cysteine residues (existing or introduced bymutation), impeding protein chain mobility that might be required forthe occurrence of an effective toxin-receptor protein binding event.

As used herein, the term “individual testing” refers to an assay,usually an insect diet bioassay, where a pesticidal protein toxin or adisabled protein toxin is tested for pesticidal activity by itself, withno other test compounds included. The term “combination testing”, refersto compounds in a similar assay but where more than one compound ispresent in the test medium or diet, in a combined mixture, as when anpesticidal protein toxin is tested in the presence of a molar excess ofa disabled protein toxin, to determine if the tested pesticidal proteintoxin binds to the same pest receptor protein (or has the same mode ofaction) as the fully functional isoform toxin from which the disabledtoxin was derived.

The term “three-domain toxins” typically refers to the core toxin,following proteolytic removal of a protoxin segment from parasporalcrystalline or Cry proteins produced by B. thuringiensis. These coretoxins display folding patterns that typically comprise three distinctstructural domains. The Domain I segment, can usually undergo variousstructural changes, following toxin binding to an insect protein, inorder to form a pore which permeates insect cells. The Domain II andDomain III segments are usually involved in the recognition and bindingof the toxin to one or more insect protein receptors, which can initiatevarious structural changes in the Domain I segment. Subsequentoligomerization of the Domain 1 segment results in formation ofmultimeric ion conducting pores which permeate insect cells, causinglysis and eventually death. Examples of core Cry toxins with establishedthree-domain crystal structures include Cry1Aa1, Cry2Aa1, Cry3Aa1,Cry3Bb1, Cry4Aa, Cry4Ba and Cry8Ea1 (deMaagd, et al, (2003) Annu. Rev.Genet. 37: 409-433).

The term “β-pore-forming toxin” refers to an insecticidal protein of theClostridium epsilon toxin ETX/Bacillus mosquitocidal toxin MTX2(ETX_MTX2) family, PF03318, related to the aerolysin protein family,PF01117. The ETX_MTX2 protein toxins contain amphipathic β-hairpin loopswhich, upon activation, are predicted to form a complex β-barrelstructure that is capable of inserting into the insect gut cell membraneand cause mortality.

Reference to pesticidal or insecticidal protein toxin activities whichare “suppressed partially or fully” in the presence of a compatibledisabled protein toxin, means that the pesticidal or insecticidal toxinactivity, measured in vitro or in vivo, is reduced in the presence of acompatible disabled protein toxin by 20% to 100%, including 20%, or 30%,or 40%, or 50%, or 60%, or 70%, or 80%, or 90%, or 100%.

As used herein, the term “cognate receptor” or “normal receptor” or“natural receptor” means any receptor, in or on or expressed by a targetpest or insect to which a pesticidal or insecticidal protein toxin bindsin order to function as an insecticide or pesticide.

Disabled protein toxins are considered to be “compatible” withpesticidal or insecticidal protein toxins when both toxins (disabled andfully-functional pesticidal) bind to the same receptor, expressed by oneor more target pests. Thus, when a disabled protein toxin binds to atarget pest receptor, any compatible pesticidal or insecticidal proteintoxin will be blocked from binding to or occupying the same receptor,resulting in suppression or reduction in the toxic efficacy of theprotein toxin on that target pest or any target pest expressing areceptor to which the compatible disabled protein toxin binds.

The novel methods described herein rely on modifying the amino acidsequence of a first pesticidal protein toxin (referred to hereininterchangeably as a fully functional polypeptide, i.e. an “FFPP”, or asa fully functional toxin, i.e. a “FFT” or the parental or native toxin)that is toxic to a target pest, resulting in a disabled protein ordisabled toxin (referred to interchangeably herein as a DP (disabledprotein), a DT (disabled toxin), or as a DIP (a disabled insecticidalprotein)), a protein that no longer exhibits the ability to causemorbidity or mortality to the target pest in the same way as the FFPP,yet the modified protein, the DT, retains the FFPP's receptor bindingactivity in the target pest. Effectively, the capacity or ability of theprotein to exhibit a toxic effect, or to cause morbidity or mortality,has been restricted. By restricted, it is also intended that the termseliminated, disabled, inactivated, inhibited, and/or removed be usedinterchangeably. Such DP's thus are intended to lack the toxic effectswhen ingested by the target pest species that are associated with theDP's cognate toxin protein from which it has been derived. Thus, a FFTfrom which a DP may be constructed retains the full natural ability tobind to a receptor in a target pest, and to cause morbidity or mortalityin the target pest, yet the disabled form of the protein, for thepurposes of the invention described herein, will be inhibited or alteredwith respect to these functions, i.e. the disabled protein will nolonger be able to bind to a receptor in the target pest and will nolonger be able to cause morbidity or mortality in the target pest. Byuse of the term “bind to”, it is intended that the terms “affinity”,“capacity”, “activation”, “structural transition”, “aggregation”,“oligomerization”, and “pore formation” be used interchangeably.

Herskowitz (Nature 329:219-222 (1987)) introduced the dominant negativeconcept and defined that “a dominant negative mutant protein will retainan intact, functional subset of the domains of the parent, wild-typeprotein, but have the complement of this subset either missing oraltered so as to be non-functional”. Interactions between functional anddysfunctional proteins can be the result of (i) differential rates ofactivation of one versus the other, (ii) competition between the two fora common receptor, (iii) disruption of oligomerization into a structurethat is no longer capable of forming functional pores, and (iv) failureto properly form pores across the membrane with which the proteins haveinteracted. Given that three-domain Cry proteins form oligomers, aninactive variant capable of interacting with the parent protein will beinhibitory as it causes the formation of non-functional oligomers.Rodriguez-Almazán et al. (PloS ONE 4(5):e5545) reported that theCry1Ab[E129K/D136N] variant acted as a “dominant negative” variant,inhibiting Cry1Ab activity towards Manduca sexta (tobacco hornworm) viaoligomerization with native Cry1Ab monomers, resulting in a loss of ionchannel or pore-forming activity of the mixed oligomer. Herskowitz alsoindicated that a monomeric protein deficient in oligomerization can alsobe inhibitory if there is limiting amount of substrate. Bt receptors,which are key in conferring the spectrum of insecticidal activity tothree-domain Cry proteins, are displayed on the midgut epithelium andare generally less abundant than the insecticidal proteins used.Herskowitz as well as Rodriguez-Almazán et al. disclosed the features ofhomologous inhibition, in which a monomeric DT variant that is deficientin self-oligomerization, but which otherwise has unaltered receptorbinding domain(s), would compete against its native counterpart on atarget insect if it is mixed with the FFPP in large excess. No prior arthas disclosed that a DT variant, when mixed with a heterologousinsecticidal protein FFPP that shares receptor(s) with the DT, wouldreduce the insecticidal activity in a dose dependent manner due to theensuing receptor competition between FFPP and DT proteins, nor did theprior art recognize that the absence of inhibition of a heterologoustoxin by such a DT means that the toxin from which the DT was derived iscompatible for use in a composition with the heterologous toxin forcontrolling the target pest to which both toxins are toxic, i.e.,neither of the two toxins are binding a common receptor, therefore bothare compatible with each other for use in a toxin composition fortargeting a single target pest for control. Methods described here relyon the use of insecticidal proteins with amino acid substitutions inparts of the protein other than those that are engaged in receptorbinding, resulting in the insecticidal protein becoming inactive,presumably due to impairment of ion channel activity, which could be aresult of any of the steps (i), (iii), or (iv) above. The DT'sexemplified in this application suppressed in vivo the insecticidalactivity of the respective FFPP from which the DT was derived in aconcentration-dependent manner, presumably because the DT retains theindependent receptor binding specificity associated with the cognateFFPP.

The methods described herein, and the resulting combinations ofpesticidal proteins described herein, are designed to be more effectiveand efficient than the methods provided in the prior art fordistinguishing compatible toxins for use in a single composition or in asingle plant, i.e. for selecting combinations of toxin proteins that canbe used together to control a single target pest using at least twodifferent independent modes of action. The method provides for a certaintoxin protein (a fully functional toxin “FFPP”) to be modified in a waythat inactivates or disables the toxic properties of the protein, i.e.the ability of the modified protein toxin to induce any toxic effects(morbidity or mortality) has been eliminated or substantially reducedcompared to the FFPP (each a disabled toxin or “DP”), yet the modifiedtoxin protein (the DP) continues to be fully capable and able tosuccessfully compete with a fully functional isoform toxin (i.e. the“FFPP”) from which the DP has been derived. The DP and the FFPP withwhich the DP competes will inhibit the binding to and block a naturaltarget receptor binding site, provided that the FFPP and the DP eachrecognize that receptor as a natural receptor in the target pest beingevaluated, or may inhibit activation, structural transition,aggregation, oligomerization, and/or pore formation, collectivelyreferred to herein as the steps leading to pesticidal activity. Byreference to “pesticidal activity”, it is intended that this be usedinterchangeably with the terms “toxicity” or “toxic properties”. Withoutintending to be bound by any one theory, it is believed that when testedin a pest bioassay, including an insect pest bioassay, a mixturecontaining the fully functional isoform toxin (fully functionalpesticidal protein, i.e. FFPP) which has demonstrated morbidity ormortality when provided in the diet of the pest, will exhibit adiminished or eliminated morbidity or mortality when a sample containinga disabled proteins (DP) is also presented in the diet, particularlywhen the DP is present in a molar excess compared to the FFPP,effectively suppressing the pesticidal activity of the fully functionalisoform toxin (FFPP), by competing for the same receptor within thetarget pest. Thus, disabled proteins, as described herein, provide auseful tool for distinguishing toxins that exhibit toxic effects againsta common target pest that confer the toxins' activity by different orcommon modes of action. Toxins that act with the same or substantiallysame mode of action are quickly identified because a DP made from afirst toxin (first FFPP) that binds to a first receptor that isrecognized by a second toxin (second FFPP), i.e. the first FFPP and thesecond FFPP commonly recognize the first receptor in the same pest thusa DP made using the first toxin (first FFPP) would effectively competewith the ability of the second FFPP to bind and exert its toxic effectsupon the pest. Therefore, the first and second toxins would not becompatible for use in a composition for controlling a target pest towhich both the first and the second toxins are each effective inconferring morbidity or mortality upon said pest. Such toxins areincompatible with each other.

Disabled protein toxins are considered to be “incompatible” withfully-functional pesticidal or insecticidal protein toxins when theybind to different receptors in a target pest. Thus when bothincompatible toxins (disabled and fully-functional pesticidal) contact atarget pest, there is no suppression or reduction in the toxic efficacyof the protein toxin on the target pest.

An important strategy in overcoming the development of insect resistanceto lethal transgenic protein toxins in crop plants, such as insecticidalBt toxins and the like, is to provide one or more additional transgenicnucleotides in the plant, expressing insecticidal protein toxins thatbind to target insect receptors that are different in comparison toinsect receptors used by other transgenic protein toxins, which aresimultaneously co-expressed in the same plant. In another embodimentdisabled protein toxins can be used to identify fully-functionaleffective toxins that are incompatible or compatible with the disabledprotein toxin. These results can be used to develop combinations oftransgenic nucleotides encoding compatible protein toxins andincompatible toxins, to provide a transgenic plant that is lethal totarget insects by using two or more modes of action, thus preventing orreducing the development of insect resistance through mutations in asingle target pest receptor.

Designing a disabled insecticidal protein toxin includes, but is notlimited to, identifying relevant residues to modify. For example, in thecase of a three-domain Cry toxin, the preference would be to modifyresidues in Domain I, especially those associated with aggregationand/or membrane piercing pore formation. This step would be followed bycloning, expressing and testing the disabled protein to identify thosepossessing no toxic effects in the target insect, compared to thefully-functional parent protein toxin, and which, when combined with acompatible toxin at a molar excess in a diet bioassay, suppresses thetoxic effects of the compatible toxin.

The elucidation of the atomic structure of compatible toxins can also beused to guide and complement approaches for selecting amino acidresidues to modify for engineering of a disabled insecticidal proteintoxin.

To generate variant proteins, an isolated nucleic acid molecule encodinga variant protein can be created by introducing one or more nucleotidesubstitutions, additions or deletions into the nucleotide sequence ofany protein or peptide, such that one or more amino acid residuesubstitutions, additions or deletions are introduced into the encodedprotein. Mutations can be introduced by standard techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis.

The preparation of sequence variants of the disabled insecticidalprotein toxin-encoding nucleic acid segments using site-directedmutagenesis is provided as a means of producing potentially usefuldisabled toxin species and is not meant to be limiting as there areother ways in which sequence variants of peptides and the DNA sequencesencoding them may be obtained or constructed.

All known Bt insecticidal Cry toxins are proteolytically activated toform soluble globular proteins that bind to insect gut cell receptorsthen undergo oligomerization and conformational transition into complextransmembrane pore assemblies. Since this transformation involves agreat deal of backbone rearrangement it is reasonable to expect thatpore formation can be arrested by placing restraints on the mobility ofcertain structural elements. It has been reported that introducingdisulfide bridges into Domain I of Cry1Aa resulted in an inactive toxinin the oxidized state, unable to form functional ion channels in planarlipid bilayers (Schwartz, et al. (1997) FEBS Letters 410, 397-402).While direct disulphide crosslinking may be feasible for in vitroexperiments, the insect gut environment may provide enough reducingpower to reduce S—S bonds and re-activate the inactive crosslinkedtoxin. In a number of the disabled insecticidal proteins describedherein, mobile elements in parent toxins are chemically cross-linked byfirst introducing cysteine residues in proximal positions that are toofar for direct disulphide formation but are close enough to becrosslinked with sulfhydryl-reactive homo-bifunctional reagents. Forexample, irreversible crosslinks can be formed between cysteines withiodoacetamide containing reagents (e.g., N,N′-ethylene bis(iodoacetamide)) or maleimide containing reagents (e.g., bis (maleimido)ethane) or similar functional crosslinkers. Creation of disabled proteintoxins using this method entails selection of two suitable residues(positioned ˜8-10 Å apart), substituting them both with cysteineresidues, expressing the resulting recombinant toxin and cross-linkingthe toxin with a bifunctional linking reagent as described above. Thismethod is particularly applicable to toxins with either known crystalstructures or where a good quality homology model can be built based ona structure of a closely related family member.

In certain embodiments, disabled protein toxins can be expressed withrecombinant DNA constructs in which a polynucleotide molecule with theopen reading frame encoding the protein is operably linked to elementssuch as a promoter and any other regulatory elements functional forexpression in the system for which the construct is intended. Forexample, plant-functional promoters can be operably linked to thedisabled protein toxin encoding sequences for expression of the proteinin plants and Bacillus thuringiensis functional promoters can beoperably linked to the disabled insecticidal protein toxin encodingsequences for expression of the protein in B. thuringiensis. Otheruseful elements that can be operably linked to the disabled proteintoxin encoding sequences include, but are not limited to, enhancers,introns, leaders, encoded protein immobilization tags (e.g., HIS-tag),encoded sub-cellular translocation peptides (e.g., plastid transitpeptides, signal peptides), encoded polypeptide sites forpost-translational modifying enzymes, ribosomal binding sites, and thelike.

An embodiment of the invention includes recombinant polynucleotidecompositions that encode disabled protein toxins, such as those setforth in SEQ ID NOs: 3, 5, 7, 11, 15, 21, 23, 25, 27, 29, 35, 39, 43,47, 51, 55, 59 and 63 encoding amino acid sequences set forth in SEQ IDNOs: 4, 6, 8, 12, 16, 22, 24, 26, 28, 30, 36, 40, 48, 52, 56, 60, and 64respectively.

Examples of methods for testing and selecting disabled protein toxinsinclude administering varying amounts of an insecticidal protein toxinand a disabled protein toxin in a diet to a target insect pest undercontrolled assay conditions (e.g., using molar ratios varying from 1:0to 1:100, respectively). Results are evaluated by measuring andcomparing the toxic potency of the fully-functional insecticidal proteintoxin in the presence and absence of the disabled protein toxin. Astatistically robust concentration-response value used for comparisonwould be the disabled protein toxin concentration which suppresses theinsecticidal toxin effect (e.g., mortality, stunting) by 50% (inhibitoryconcentration or IC₅₀).

EXAMPLES

In view of the foregoing, those of skill in the art should appreciatethat changes can be made in the specific aspects which are disclosed andstill obtain a like or similar result without departing from the spiritand scope of the invention. Thus, specific structural and functionaldetails disclosed herein are not to be interpreted as limiting. Itshould be understood that the entire disclosure of each patent, patentapplication, and publication referenced herein are incorporated hereinby reference in their entirety.

Example 1

This example illustrates methods for producing a disabled toxin (DT)from a fully functional pesticidal protein (FFPP) by starting with anexemplary toxin protein, a Bt insecticidal pore forming protein(Cry1Ab), consisting of the amino acid sequence as set forth in SEQ IDNO:2.

Methods are known in the art for introducing changes into the primarystructure of a protein.

Site-directed (also referred to as “site-specific”) mutagenesis was usedto introduce coding sequence modifications to a nucleic acid sequenceencoding a Cry1Ab toxin, a FFPP that, when provided in the diet oftarget insect pest larvae, is able to cause morbidity and/or mortalityto such target insect pests. The nucleic acid sequence modifications aredesigned to result in the construction of a nucleotide coding sequenceencoding one or more amino acid sequence variations within the Cry1Abamino acid sequence, decreasing or eliminating the variant proteins'ability to form pores, to aggregate together with other Cry1Ab toxinmolecules, into a pore complex, and therefore lacking the ability tocause morbidity and/or mortality to target insects in the same way asthe unmodified FFPP Cry1Ab. The disabled Cry1Ab toxin (DT) is no longerable to exhibit any substantial toxic effect when provided in the dietat high concentrations to the applicable target pest, but isdemonstrated to be competitive with the unmodified FFPP from which theDT was formed, likely by binding at the same receptor binding site andinhibiting binding of the unmodified FFPP. It is preferred that themodifications to an FFPP that is a characteristic Cry-class proteinexhibiting a typical three-dimensional structure having domains I, II,and III, be limited to amino acids within the domain I architecture ofthe three domain Cry toxin because this domain is typically the segmentthat is primarily responsible for membrane penetration and poreformation. Domains II and III of such three domain toxins are involvedin insect receptor binding and the present invention is intended toavoid disrupting the receptor binding amino acid segments of FFPPprotein toxins regardless of their three-dimensional architecture. Theexamples described herein illustrate modifications that produce disabledtoxins (DT's), and the use of such disabled toxins to confirm (i) anyDT's interference with binding of the unmodified FFPP from which the DTis derived; (ii) any DT's interference with certain other and unmodified(i.e., different) FFPP's other than that from which the DT is derived,which then illustrate the overlap of binding of such other differentFFPP's with the unmodified FFPP from which the DT is derived,illustrating the practicality of avoiding combinations of such FFPP'sthat exhibit such overlapping binding characteristics with theunmodified FFPP from which the DT is derived; and (iii) the absence ofinterference with the binding of certain other FFPP's that are differentfrom the unmodified FFPP from which the DT is derived, illustrating thespecific different FFPP's which should be considered useful forcombinations with the unmodified FFPP from which the DT is derived.These are the bases for the utility of the present invention. The rapididentification of those different FFPP's that are compatible for usewith the unmodified FFPP from which the DT is derived in compositionsand in plants to control an applicable single target pest that issusceptible to two or more different FFPP's in a single commercialembodiment. The conservative nature of the proteins within the Cry1class of toxin proteins makes the changes are shown here as having beenintroduced into the Cry1Ab amino acid sequence as set forth in SEQ IDNO:4, 6 and 8 (when each are compared to the unmodified Cry1Ab aminoacid sequence as set forth in SEQ ID NO:2) effective when introducedinto other closely related Cry1's having these amino acid residues atthese positions, and guides the person of skill in the art tounderstanding the positions in different toxin proteins that may resultin similar effects when such modifications are introduced into othersequence-related proteins including chimeras containing Cry1A relateddomain I segments.

Crosslinking cysteine residues that are artificially introduced intoamino acid segments as supplementary amino acids or as substitutionsinto positions within the FFPP toxin protein amino acid sequence canresult in inactivation of the ability of the modified toxin to confertoxicity, provided that such cysteine insertions or substitutions do notinterfere with the modified toxin's ability to bind to the applicablereceptor in the gut of the target insect to which the unmodified FFPPalso binds. Bt insecticidal toxins undergo oligomerization andconformational transitions into transmembrane pore assemblies followingbinding to specific insect gut receptors. Therefore, pore formation canbe prevented by placing restraints on the mobility of structuralelements in the pore-forming components of the toxin protein. Asexemplified herein, mobile elements in a toxin that are responsible forpore formation, and thus the toxic features of the pesticidal protein,can be impeded by introducing cysteine residues in the toxin proteinamino acid sequence, so that each such cysteine substitution orinsertion is too far apart in three dimensional conformational space toallow for direct disulphide formation, but which are within a proximityto each other in three dimensional space within the architecture of themodified toxin protein to be irreversibly crosslinked withhomobifunctional sulfhydryl-reactive crosslinking reagents, such asiodoacetamides (e.g., N,N′-ethylenebis(iodoacetamide)) or maleimides(e.g., bis(maleimido) ethane) or similar functional crosslinkers. Suchcrosslinking will cause a significant if not total loss of the abilityof the protein to exhibit any toxic effects, but will not diminish themodified proteins' (DT) ability to bind to the cognate receptor to whichthe unmodified FFPP from which the modified protein (DT) has beenderived is also able to bind. Creation of disabled toxins using thismethod entails selection of two suitable residues (typically positionedat least about 8-10 Å apart), introducing changes into the nucleotidesequence encoding the toxin that results in a cysteine amino acid to besubstituted for the normal amino acid at these spaced residue positions,expressing the modified protein containing the cysteine residues, andcross-linking the cysteines within the individual proteins expressedfrom the modified coding sequence with a homo-bifunctional reagent,similar to those described above. The modified FFPP amino acid sequencevariant toxin will be able to bind to the cognate receptor but not causetoxic pore formation, and can be demonstrated to compete for receptorbinding with the unmodified FFPP form of the toxin from which thecysteine modified disabled toxin (DT) has been derived.

The disabled Cry1Ab3-DIP3 protein containing the amino acid sequencevariant residues I109C/D129C in helices 3 and 4 of domain 1 wascompletely inactive towards multiple lepidopteran species in the absenceof any cross-linking. The crystal structure of the protein providedevidence that the two cysteine residues are not oxidized to a disulfidebridge, but rather they comprise free sulfhydryl groups in theI109C/D129C variant. In addition, we also found that both the soluble,pre-proteolyzed form of this toxin and its precursor crystal/sporepreparation were disabled. The fact that the crystal/spore preparationof the native Cry1Ab has insecticidal activity on these targetlepidopteran pests indicates that the insect lumen in these pests has aphysiological environment sufficient to reduce multiple disulfidebridges interconnecting the protoxins in the crystalline form. Thus, itis unlikely that this protein would re-oxidize between helices 3 & 4 inthe lumen following ingestion. A more plausible disabling mechanism isthe disruption of self-oligomerization. Numerous contributions arealready published on elucidating the function of helix 3 and 4 of Cry1Aproteins by characterizing single point mutants, and these studiessuggest that some of the helix 3 and 4 positions are critical forinsecticidal activity and pore-formation. Vachon and Girard et al.(Vachon, Prefontaine et al. 2004, Girard, Vachon et al. 2008, Girard,Vachon et al. 2009) showed that cysteine mutagenesis of the helix 4 E129position resulted in loss of bioassay activity as well as loss ofpore-formation (osmotic shock) in brush border membrane. The same groupcommunicated that E129C has unaltered BBMV binding inferred from thepore-formation assay set up as competition between wild-type toxin andvariant. Because their competitive binding assay is set up with apore-formation assay read-out, the results cannot readily distinguishbetween competition binding and oligomer poisoning. Additional insightsto the same position were published by Rodriguez-Almazán et al.(Rodriguez-Almazán C 2009) showing that the E129K variant is inhibitingits native counterpart via oligomer poisoning. Regarding the helix 3positions, Jimenez-Juarez et al. (Jimenez-Juarez, Munoz-Garay et al.2007) reported on a helix 3 mutagenesis study that identified twovariants, R99E (which was used as a control in this application) andY107E, that lost its insecticidal activity, and were characterized asnon-functional oligomers with reduced stability. Taken together, thehypothesis could be that helix 3 and 4 comprise an extensive surface forself-oligomerization that is fully disrupted upon stacking key mutationsin helix 3 and 4. Single point mutants showed partial reduction ofself-oligomerization and therefore a concomitant oligomer poisoningdominant negative effect in competition assays; thus, these probes arenot ideal if one wants to observe receptor utilization in isolation. Ourstudies suggest that the I109C/D129C variant residues in Cry1Abcompletely disrupt the oligomerization step, given that (i) a largeexcess of the disabled toxin (DT) competitor was required to inhibit thehomologous native protein, and (ii) Cry1A.105 normally insecticidalprotein was not competed by a Cry1A.1088 disabled toxin even thoughthese are both chimeric proteins that share the same domain 1, whosefunction is associated with both the oligomerization and pore-formationsteps.

Initial studies with Cry1Ab3 variants demonstrated that Cry1Ab3-DIP3,containing the I109C/E129C substitutions, satisfied criteria for use asa disabled toxin (DT) in mode of action studies. The disabled proteinCry1Ab3-DIP3 exhibited 1) no significant insecticidal activity towardsany of the lepidopteran species tested, 2) no detectable ion channelactivity in planar lipid bilayer experiments, 3) no apparent differencesin susceptibility to processing with trypsin, 4) no significantcompetition in bioassays with the native Cry1Ab3 protein at molarconcentrations of 1:1, and 5) significant competition with Cry1Ab3 infeeding assays with multiple lepidopteran species when presented in amolar excess of ≥10. Crosslinking of the engineered cysteine residueswith EBI was not required to inactivate the protein, a feature thatprovides additional uses for the protein as discussed below. This methodenables the skilled artisan to classify Cry proteins or otherinsecticidal proteins into groups that are likely to share receptorbinding sites and to prioritize insecticidal proteins that appear tooperate via an independent mode of action, as evidenced by the absenceof any competition in insect bioassays. Finally, because the method onlyrequires a validated insect bioassay, it is possible to assesscompetition quickly across a wide range of insect target species.

Example 2

This example illustrates the competition for receptor binding between anunmodified FFPP Cry1Ab toxin and several Cry1Ab amino acid sequencevariants (each a different DT) that are each demonstrated to be unableto exert toxic effects upon the target insect species, and which areeach unimpaired from binding to the receptor to which the unmodifiedFFPP also binds.

Three different DT's, each a Cry1Ab disabled toxin, were generated froman unmodified FFPP Cry1Ab amino acid sequence as set forth in SEQ IDNO:2, and each shown to compete with the unmodified Cry1Ab in bioassaysusing three different insect species, each shown also to be susceptibleto unmodified Cry1Ab. Disabled toxins Cry1Ab_1 (DT1 having the aminoacid sequence as set forth in SEQ ID NO:4) and Cry1Ab_2 (DT2 having theamino acid sequence as set forth in SEQ ID NO:6) were produced byintroducing amino acid sequence changes in Domain I of the FFPP Cry1Abtoxin amino acid sequence set forth in SEQ ID NO:2, and the changesintroduced are shown in Table 1. DT1 contains a single amino acidsubstitution, R99E, which results in disabling the toxic properties ofthe FFPP Cry1Ab protein in applicable target pest species. DT2 containstwo different amino acid substitutions, E129K and D136N, togetherresulting in the disabling of the toxic properties of the FFPP Cry1Abprotein in applicable target pest species. Cry1Ab_3 (DT3 having theamino acid sequence as set forth in SEQ ID NO:8) was produced bysubstitution of two spatially separated amino acids in the amino acidsequence of Cry1Ab as set forth in SEQ ID NO:2 with cysteine residues,using the method as described above in Example 1. A cysteine residue wassubstituted for isoleucine at amino acid position 109 and a cysteinesubstituted for glutamate at position 129 within the primary amino acidsequence of Cry1Ab as set forth in SEQ ID NO:2. Variant Cry1Ab3 (DT3),was designed so that the cysteine residues are surface exposed and thencapable of being crosslinked with homo-bifunctional reducing agents asdescribed above in Example 1.

TABLE 1 Toxicity Disabling Cry1Ab Modifications Amino Acid Sequence SetForth in Cry SEQ ID Amino Acid protein Alias NO: Changes Protein typeCry1Ab FFPP 2 — Unmodified Cry1Ab toxin Cry1Ab_1 DT1 4 R99E DisabledCry1Ab toxin Cry1Ab_2 DT2 6 E129K, D136N Disabled Cry1Ab toxin Cry1Ab_3DT3 8 I109C, E129C Disabled Cry1Ab toxin

Insect bioassays were conducted using the toxic and unmodified form ofFFPP Cry1Ab (SEQ ID NO:2) and the disabled Cry1Ab modified proteins(DT1, DT2, and DT3) having the amino acid sequences as set forthrespectively in SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8) to establishconcentrations of unmodified Cry1Ab required to titrate the mortalitycurve for each target insect species tested and to confirm that each DTwas devoid or substantially devoid of any pesticidal activity in theapplicable target insect species when tested at high concentrations.Subsequently, the unmodified Cry1Ab (FFPP) protein was mixed with eachdisabled toxin at molar ratios of 1:1 and 1:10 and tested in insectbioassay to determine the extent to which the disabled toxin (DT)suppresses the unmodified FFPP Cry1Ab. Table 2 shows the results of abioassay using Manduca sexta (Tobacco hornworm, THW) as the targetinsect species. In this example, 0.04 ppm samples of unmodified FFPPCry1Ab resulted in severe stunting of the larvae while 4 ppm samples ofeach disabled toxin tested alone exhibited no significant activity (a100-fold increase in concentration of the DT compared to the unmodifiedtoxin). When each DT was combined in a 1:1 molar ratio with unmodifiedFFPP Cry1Ab, little or no effect was observed on unmodified Cry1Abactivity, although the unmodified Cry1Ab+DT2 mixture was less activethan unmodified Cry1Ab when tested in the absence of DT2. When each DTwas tested at a 10 fold excess compared to the amount of unmodified FFPPCry1Ab, both DT2 and DT3 each independently suppressed the activity ofCry1Ab, illustrating that the disabled toxins DT2 and DT3 were each ableto compete for the unmodified FFPP Cry1Ab receptor without causing anymortality or morbidity.

DT1 was less effective in suppressing the activity of unmodified FFPPCry1Ab when presented in ten-fold molar excess, but the activity of themixture was notably lower than that of the activity when unmodified FFPPCry1Ab was tested alone. Similar results were obtained using Heliothisvirescens (tobacco budworm, TBW) as the target pest species (datapresented in Table 3). In this assay, the stunting data resultsdemonstrate that (1) DT2 and DT3 show little or no activity against TBW,(2) neither DT2 nor DT3 impacts the activity of unmodified FFPP Cry1Abwhen presented in a 1:1 molar ratio, and 3) both DT2 and DT3 suppressthe activity of unmodified FFPP Cry1Ab when each DT is presented in theassay in a ten fold molar excess. These results are consistent withthose obtained using Manduca sexta as the target pest species. However,in this assay the disabled toxin DT1 displayed significant stuntingeffects in TBW at the concentration tested and thus could not be used tosuppress the activity of unmodified FFPP Cry1Ab toxin.

TABLE 2 Disabled Cry1Ab Competes with Unmodified Cry1Ab for ReceptorBinding in THW Homologous Competition Against Cry1Ab in THW SEQ ID NO:/Active on 1:1 Cry Protein THW 1:10 2/FFPP Cry1Ab + NA 4/DT1 − − − 6/DT2− + + 8/DT3 − − +

Ostrinia nubilalis (European corn borer, ECB) was also tested as atarget pest species and the results are shown in Table 3. The datasummarized in Table 3 indicate that 1) each of the different DT'sexhibit no significant toxic activity when tested against ECB at 50 ppm,2) the three DT's have no significant impact on the activity ofunmodified FFPP Cry1Ab when presented at a 1:1 molar ratio, and 3) allthree disabled toxins suppress the activity of unmodified FFPP Cry1Abwhen each are present at a 10 fold molar excess.

TABLE 3 Disabled Cry1Ab Competes with Unmodified FFPP Cry1Ab forReceptor Binding in Target Pests TBW and ECB Homologous CompetitionAgainst Homologous Cry1Ab in ECB SEQ ID No:/ Competition Against Activeon 1:1 Cry Protein Cry1Ab in TBW ECB 1:10 2/Cry1Ab NA + NA 4/DT1 NA −− + 6/DT2 + − − + 8/DT3 + − +/− +

Example 3

This example illustrates that identifying that a first DT derived from afirst unmodified FFPP that competes with a second unmodified FFPPdifferent from the first, and that a second DT derived from the secondunmodified FFPP competes with the first unmodified FFPP, isdeterminative that the first and second unmodified FFPP's are notcompatible for use together in an insect resistance management system,i.e., the two unmodified FFPP's are likely to be capable of binding tothe same or substantially similar receptors in an applicable target pestspecies, and therefore are not candidates for use together in acomposition for controlling the pest, even though both toxins may beeffective at controlling the target pest. This is because the likelihoodof development of resistance to one of the toxins is high, and thedevelopment of resistance to one of the toxins would likely be effectivein reducing or eliminating the other toxin's ability to control the sametarget pest.

In this example, two different lepidopteran toxic chimeric FFPPproteins, a TIC105 (composed of Domain I and II of Cry1Ab and Domain IIIof Cry1Fa and having the amino acid sequence as set forth in SEQ ID NO:10) and TIC107 (composed of Cry1Ab domains I and II and Cry1Ac DomainIII and having the amino acid sequence as set forth in SEQ ID NO: 14)are each used separately to derive disabled toxin amino acid sequencevariants (DT's) that rely on the incorporation of two cysteine residueswithin Domain I of each of these different chimeric FFPP's, substitutingeach of isoleucine at position 109 and glutamate at position 129 with acysteine. The disabled toxin amino acid sequences for each of these DTproteins are set forth in SEQ ID NO:12 (modified TIC105, DT4) and SEQ IDNO: 16 (modified TIC107, DT5). The disabled toxins DT4 and DT5 were eachtested for insect toxicity and competition with their respective FFPP'sfrom which they were each derived, against the target pest speciesDiatraea grandiosella (southwestern corn borer, SWCB), Helicoverpa zea(corn earworm, CEW) and Spodoptera frugiperda (fall armyworm, FAW),which are each known to be sensitive to each of the unmodified FFPP'sTIC105 and TIC107. Similar to the examples above, the molar ratios usedto determine efficacy of the system for making and observing thesebinding comparisons was established by titration with increasingconcentrations of the disabled proteins. Concentrations of 1:1 and 1:20molar excess of each of the DT's was used for making comparisons whentesting with SWBC and with FAW, and a 1:80 molar excess was required formaking the comparisons when testing in CEW, as complete suppression ofmortality by the DT's was not observed unless the DT was included at thehigher concentration.

The results of testing the unmodified chimeric toxins alone and togetherwith each of the respective DT's are shown in Tables 4 and 5.

TABLE 4 Disabled Chimeric Toxins Are Competitive with UnmodifiedChimeric Toxins When Tested in SWCB Homologous Competition Against FFPPToxin in SWCB SEQ ID NO:/ Amino Acid Active on 1:1 Cry ProteinSubstitutions SWCB 1:20 10/TIC105 — + NA 12/DT4 I109C, E129C − +/− +14/TIC107 — + NA 16/DT5 I109C, E129C − − +

TABLE 5 Disabled Chimeric Toxins Compete with Unmodified Toxins forReceptor Binding in CEW and FAW Homologous Homologous CompetitionCompetition Against Parent Against Parent Toxin in CEW Toxin in FAW SEQID No:/ Active on 1:1 Active on 1:1 Cry Protein CEW 1:80 FAW 1:2010/TIC105 + NA + NA 12/DT4 − − − − + + 14/TIC107 + NA + NA 16/DT5 − − −− + +

Both disabled toxins, DT4 and DT5, exhibit strong homologous competitionwith their respective FFPP's from which these were derived in the SWCBand FAW assays, with a compete suppression of mortality observed at onlytwenty-fold molar excess of the disabled toxin. However, against CEW,DT4 exhibits only partial homologous competition at eighty-fold molarexcess, as mortality was observed at a twenty-fold molar excess. Incontrast, DT5 exhibits strong homologous competition in the CEW assay.These results suggest that the DT's have a diminished affinity for thesame receptor that the respective chimeric protein is targeting forbinding, or possibly that there is more than one receptor being targetedfor binding and that the disablement of the respective toxin has onlypartially inhibited the binding of the toxin to one or to both.

Example 4

This example teaches heterologous competition between a first disabledtoxin 1-DT derived from a first FFPP (1-FFPP) and a second FFPP (2-FFPP)different from the first FFPP by at least one amino acid, wherein both1-FFPP and 2-FFPP are each toxic to the same target pest. The exampleteaches specifically the testing for efficacy of the three disabledtoxins described in Example 2 (DT1, DT2, and DT3) in suppressing theinsecticidal activity of toxins that are not the same as the unmodifiedFFPP Cry1Ab from which the three disabled toxins were derived.

Specifically tested were two FFPP's that are different from Cry1Ab.Cry1Ab in this Example 4 is referred to as 1-FFPP, and has the aminoacid sequence as set forth in SEQ ID NO:2). TIC105, referred to in thisExample 4 as 2-FFPP, has the amino acid sequence as set forth in SEQ IDNO: 10. Cry2Ab, referred to in this Example 4 as 3-FFPP, has the aminoacid sequence as set forth in SEQ ID NO:18. TIC105 is a chimeric Cry1Aprotein sharing significant sequence similarity with Cry1Ab across theDomains I and II segments, while Cry2Ab is known to exhibit amode-of-action that is distinct from that of Cry1Ab and neither proteinhas any significant overlap of amino acid sequence identity orsimilarity. For initial assays, a maximum concentration of disabledtoxin (50 ppm) was used in mixtures with the active unmodified FFPPtoxins.

Results from studies using THW are shown in Table 6. As expected,competition was observed when the disabled Cry1Ab proteins DT1, DT2 andDT3 were tested in assays with the unmodified FFPP Cry1Ab (1-FFPP). Nocompetition was observed when these Cry1Ab disabled toxins DT1, DT2 orDT3 were tested in bioassays in which the unmodified FFPP was Cry2Ab(3-FFPP), consistent with the view that Cry1Ab and Cry2Ab each bind todifferent target receptors.

With reference to the TIC105 data, a differential effect was observedwith the different disabled toxins. DT2 exhibited complete suppressionof TIC105 (2-FFPP) activity, while DT1 and DT3 showed no heterologouscompetition when tested against 2-FFPP. The suppression observed by DT2may be due to a “dominant negative” phenotype associated with thisdisabled toxin, an effect that may not be due to competition forreceptor binding but rather to interference with ion channel assemblyand activity (see for example, Rodriguez-Almazan et al. (2009) PloS ONE:e5545). In this case, DT2 may not be competing for TIC105 (2-FFPP)receptor binding sites, but instead is forming hetero-oligomers with theTIC105 toxin resulting in a complex that is defective in forming ionchannels or pores.

The results with tobacco budworm (TBW) are also shown in Table 6. Inthis assay, mortality was low across all treatments, so stunting datawas evaluated instead. The disabled toxins DT2 and DT3 exhibit completesuppression of Cry1Ab (1-FFPP), partial suppression of TIC105 (2-FFPP),and no suppression of Cry2Ab (3-FFPP). These results suggest a partialoverlap in the receptor binding sites of Cry1Ab and TIC105 and nooverlap between the receptor binding sites of Cry1Ab and Cry2Ab in TBW.

The stunting data with Diatraea grandiosella (southwestern corn borer;SWCB) indicate no competition between the DT's tested and Cry2Ab(3-FFPP), or when tested with TIC105 (2-FFPP) (Table 6). The disabledtoxins DT2 and DT3 were both effective in suppressing the activity ofCry1Ab (1-FFPP) in this species.

TABLE 6 Disabled Toxin Competitive Binding with Heterologous ToxinsTested in THW, TBW and SWCB SEQ ID Active Heterologous* ActiveHeterologous* Active Heterologous* No:/Cry on Competition on Competitionin on Competition in Protein THW in THW TBW TBW SWCB SWCB 2/Cry1Ab +NA + NA + NA 4/DT1 − + − − − + 6/DT2 − + − + − + 8/DT3 − + − + − +18/Cry2Ab2 + NA + NA + NA 4/DT1 − − − − − − 6/DT2 − − − − − − 8/DT3 − −− − − − 12/TIC105 + NA + NA + NA 4/DT1 − − − − − − 6/DT2 − + − + − −8/DT3 − + − + − − *Competition is homologous with respect to Cry1Ab.

Example 5 Homologous Competition Between Cry51Aa and Disabled ToxinAmino Acid Sequence Variants

This example illustrates the effect of producing disabled ß-pore formingtoxins DT6, DT7, DT8, and DT9 and testing these against the unmodifiedFFPP ß-pore forming toxin from which these are derived (a Cry51Aa toxin,having the amino acid sequence as set forth in SEQ ID NO:32, TIC834_14)for competition with at least one common receptor. SEQ ID NO:32 isillustrative of a member of the β-pore-forming insecticidal crystalprotein class and is also referenced herein as a Cry51Aa toxin (i.e.,alternatively referred to as a Cry51Aa2 or Cry51Aa2.834_14 and eachreferred to as a ßFFPP, Cry51Aa2.834_14 and TIC834_14 alternativelyreferred to herein as ßFFPP-2).

ßFFPP-2 exhibits insecticidal activity against Hemipteran insects,including Lygus species such as Lygus lineolaris (tarnished plant bug)and Lygus hesperus (western tarnished plant bug) (see U.S. PatentApplication No. 2013/0269060, in which TIC834_14 and TIC834_16 are eachreferenced).

In this Example 5, four different disabled variants of ßFFPP-2 weregenerated and are shown to compete with ßFFPP-1, a TIC843_16, having theamino acid sequence as set forth in SEQ ID NO:20, an unmodifiedinsecticidal protein that exhibits improved activity in bioassaysagainst Lygus hesperus and Lygus lineolaris. The disabled toxins and themodifications that have been introduced into these are shown in Table 7.The ßFFPP-1 and ßFFPP-2 are indistinguishable in bioassays in thisExample, even though the are not identical, i.e., the ßFFPP-2 sequencecontains an alanine at amino acid sequence position 248 and an arginineat position 270 compared to ßFFPP-1 which sequence contains a valine anda tryptophan, respectively, at these same positions.

It has been shown that β-pore-forming toxin activity involves formationof heptameric oligomers with a central pore generated from stem loopstructures (see, for example, Tanaka, Y., et al. (2011) Protein Science20, 448-456 and De, S. and Olson, R. (2011) Proc. Natl. Acad. Sci. 108,7385-7390). In these pores, the stem loops are amphipathic, antiparallelbeta-strands, with the hydrophilic side lining the pore solvent channeland the hydrophobic pore face directed toward the hydrophobic membrane.Prior structural characterization of ßFFPP (see U.S. Patent ApplicationNo. 2013/0269060, in which TIC834 is referenced as TIC807) has revealedan amphipathic beta-pore-forming loop (bPFL).

The strategy to produce disabled toxins of ßFFPP toxins ßFFPP-1 orßFFPP-2 involved selected mutagenesis and subsequent chemicalmodification centered on the bPFL to inhibit productive pore formation.Specifically, double cysteine (Cys) amino acid sequence variants ofßFFPP-2 were designed so that cysteine residues were substituted forcognate amino acids at positions within the amino acid sequence and withsufficient spacing that the Cys residues could be crosslinked covalentlywith a bifunctional reducing reagent, such as N,N′-ethylene bis(iodoacetamide). Two types of double Cys variants were designed: type(a) variants with both Cys residues within the bPFL and on thehydrophilic face, and type (b) variants with one Cys residue within thebPFL on the hydrophilic face and another within the adjoining proteinstructure.

Type (a) double Cys variants include DT8 (ßFFPP-1 modified to containthese type (a) cysteine substitutions and having the amino acid sequenceas set forth in SEQ ID NO:26) and DT9 (ßFFPP-1 modified to contain thesetype (a) cysteine substitutions and having the amino acid sequence asset forth in SEQ ID NO:28). Type (b) double Cys variants include DT6(ßFFPP-1 modified to have these type (b) cysteine substitutions andhaving the amino acid sequence as set forth in SEQ ID NO:22), and DT7(ßFFPP-1 modified to have these type (b) cysteine substitutions andhaving the amino acid sequence as set forth in SEQ ID NO:24). Thespecific amino acid positions which were modified by cysteinesubstitution are shown in Table 7 and as exemplified in the sequences asset forth in the Sequence Listing.

TABLE 7 Disabled toxin Amino Acid Sequence Variants of βFFPP Amino AcidSEQ ID Sequence Cry protein NO: Modifications Protein type βFFPP 20 —βFFPP DT6 22 D55C, S117C DT DT7 24 D55C, S131C DT DT8 26 P121C, T129C DTDT9 28 P121C, T133C DT

The four ßFFPP DT proteins DT6, DT7, DT8 and DT9 were expressed in anacrystalliferous strain of B. thuringiensis. After purification andprior to cross-linking, the DT protein samples were dissolved in 50 mMcarbonate-pH 9.2, 200 mM NaCl. A 20 mM N,N′-ethylene bis (iodoacetamide)stock solution was prepared by dissolving the reagent in DMSO, which wasadded at 10× the molar protein concentration. Mass spectral analyseswere conducted on samples, before and after reaction with thebifunctional reagent, to verify that productive crosslinking hadoccurred.

Insect diet bioassays were run individually with the unmodified ßFFPP-1protein and individually with each of the four disabled variants DT6,DT7, DT8 and DT9 to (a) establish tittered concentrations of unmodifiedßFFPP-1 required to cause significant mortality for each insect speciestested, and (b) confirm that the disabled proteins retain little or noinsecticidal activity at high concentrations. Subsequently, theunmodified ßFFPP-1 was mixed separately with each disabled toxin atthree different molar ratios (1:2, 1:20 and 1:50) in which each DT waspresent in excess, and tested in insect bioassays to determine the levelof suppression of the unmodified toxin that each disabled toxin is ableto provide. Table 8 shows the results of bioassays using the targetinsect pest Lygus lineolaris (tarnished plant bug).

TABLE 8 Disabled Toxins DT6, DT7, DT8, and DT9 Compete with UnmodifiedβFFPP-1 Toxin for Receptor Binding in Lygus lineolaris (tarnished plantbug) Homologous Competition SEQ ID NO:/ Active on Against βFFPP in CryProtein L. lineolaris L. lineolaris 20/βFFPP + NA 22/DT6 − + 24/DT7 − +26/DT8 − + 28/DT9 − +

DT7 was most effective in suppressing the activity of ßFFPP-1, whileDT6, DT8 and DT9 were less effective. Each of the crosslinked disabledvariants exhibited insignificant levels of toxicity, while samples ofthese variants that were not exposed to N,N′-ethylenebis(iodoacetamide)exhibited significant levels of toxicity illustrating that thesubstitution of the natural amino acids at the specified positions withcysteines was not significantly detrimental to the toxic pore formingproperties of these proteins. For example, the non-crosslinked versionof DT7 at 200 ppm resulted in 88% mortality, while crosslinked DT7 at2000 ppm had a negligible effect. In addition, disabled variant DT7 wasalso effective in suppressing the activity of ßFFPP-1 in diet bioassaytesting against Lygus hesperus (western tarnished plant bug). DT7 alone(crosslinked) exhibited no toxicity against L. hesperus.

These results illustrate that the unmodified ßFFPP-1 and the disabledforms of this toxin, DT6, DT7, DT8, and DT9 each bind to the same set ofreceptors, and also illustrate the speed with which a more rapid andefficient means for identifying combinations of two or more toxins thatcan be used together to control a single target pest species susceptibleto each of the toxins, i.e., toxins that do not compete for the samereceptor, and therefore provide a more durable pest control product thatis less susceptible to the development of resistance.

Example 6 Assessment of the Contribution of TIC105 and Cry2Ab to SoybeanLooper Control Using Premixed Lyophilized Soybean Leaf Tissue

This example illustrates the use of two different disabled toxinsderived from two different unmodified toxins, to demonstrate therelative contribution of each toxin to control Soybean looper(Chrysodeixis includens) using premixed lyophilized soybean leaf tissuesamples from transgenic soybean plants expressing the insect toxinsTIC105 or Cry2Ab.

Leaf tissue samples were obtained from two different transgenic soybeanevents, one expressing the insect toxin TIC105 and the other expressingCry2Ab. The tissue samples were then lyophilized into a powder and addedto an insect diet alone and in various combinations. The ratio of toxinin each sample was adjusted to provide a specific ratio of LC values,ranging from LC10 to LC90 for each toxin. The amounts of tissues to bepremixed were determined in a separate tissue dilution experiment forboth single events, and the mixtures represent a combined approximateLC95 dose. Table 9 shows the results of these combinations.

TABLE 9 LC95 Combinations of FFPP TIC105 and FFPP Cry2Ab andCorresponding Concentration of Lyophilized Soybean Event Tissue.TIC105(LC) + (X)mg/ml TIC105 + Cry2Ab(LC) (Y)mg/ml Cry2Ab TIC105(LC10) + 0.2 mg/ml TIC105 + Cry2Ab2 (LC90) 7.8 mg/ml Cry2Ab TIC105(LC30) + 0.6 mg/ml TIC105 + Cry2Ab2 (LC70) 5.6 mg/ml Cry2Ab TIC105(LC50) + 1.0 mg/ml TIC105 + Cry2Ab2 (LC50) 4.0 mg/ml Cry2Ab TIC105(LC70) + 1.4 mg/ml TIC105 + Cry2Ab2 (LC30) 2.4 mg/ml Cry2Ab TIC105(LC90) + 1.8 mg/ml TIC105 + Cry2Ab2 (LC10) 0.8 mg/ml Cry2Ab

Combinations of TIC105 and Cry2Ab that result in LC95 toxic effectsagainst Soybean looper; Column 1, purified TIC105 and Cry2Ab mixtures;Column 2, extrapolated amounts of toxin present in lyophilized powdersof soybean events expressing each of these proteins.

Increasing concentrations of the disabled toxins, DT4 or DT10separately, or both DT4 and DT9 in combinations, were presented as adiet overlay in concentrations of 0.1, 1.0, and 2.0 milligrams permilliliter (mg/ml) over the insect diet comprising the TIC105 and Cry2Ablyophilized tissue samples. First instar Soybean looper larvae wereallowed to feed on the insect diet for four days. Mortality and stuntingwas determined for each sample for each LC ratio. Loss of insecticidalactivity due to the disabled toxin competition is presented in Table 10below which shows the mean percent mortality for each LC ratio andcorresponding overlaid disabled toxin(s).

TABLE 10 Mean percent mortality of Soybean looper larvae fed TIC105 andCry2Ab in the presence of the disabled toxins, DT4 and DT10. DisabledTIC105 (LC) + Disabled Toxin Percent Cry2Ab2(LC) Toxin (mg/ml) MortalitySEM TIC105 (LC10) + 0 91.67 4.17 Cry2Ab2 (LC90) DT4 0.1 50.00 7.22 DT4 147.62 2.38 DT4 2 62.50 25.00 DT10 0.1 25.00 0.00 DT10 1 4.17 4.17 DT10 216.67 8.33 DT4 and DT10 0.1 4.17 4.17 DT4 and DT10 1 0.00 0.00 DT4 andDT10 2 0.00 0.00 TIC105 (LC30) + 0 87.50 0.00 Cry2Ab2 (LC70) DT4 0.152.98 9.91 DT4 1 50.00 7.22 DT4 2 37.50 7.22 DT10 0.1 66.67 8.33 DT10 141.67 8.33 DT10 2 12.50 7.22 DT4 and DT10 0.1 12.50 7.22 DT4 and DT10 10.00 0.00 DT4 and DT10 2 0.00 0.00 TIC105 (LC50) + 0 95.83 4.17 Cry2Ab2(LC50) DT4 0.1 87.50 7.22 DT4 1 33.33 11.02 DT4 2 12.50 7.22 DT10 0.191.67 4.17 DT10 1 87.50 7.22 DT10 2 37.50 0.00 DT4 and DT10 0.1 33.334.17 DT4 and DT10 1 0.00 0.00 DT4 and DT10 2 0.00 0.00 TIC105 (LC70) + 095.83 4.17 Cry2Ab2 (LC30) DT4 0.1 45.83 15.02 DT4 1 16.67 4.17 DT4 216.67 4.17 DT10 0.1 91.67 4.17 DT10 1 54.17 15.02 DT10 2 79.17 11.02 DT4and DT10 0.1 79.17 11.02 DT4 and DT10 1 0.00 0.00 DT4 and DT10 2 0.000.00 0 83.33 4.17 DT4 0.1 79.17 15.02 DT4 1 58.33 18.16 DT4 2 0.00 0.00TIC105 (LC90) + DT10 0.1 87.50 7.22 Cry2Ab2 (LC10) DT10 1 95.83 4.17DT10 2 70.83 15.02 DT4 and DT10 0.1 62.50 19.09 DT4 and DT10 1 16.6711.02 DT4 and DT10 2 0.00 0.00

In an earlier experiment, the disabled toxins DT4 and DT10 at one andtwo milligrams per milliliter dose, respectively, completely competed(i.e., tittered) the TIC105 and Cry2Ab single soybean tissues sampleswhen these samples were not mixed. When these tissues were premixed,complete inhibition of the combined insecticidal activity of TIC105 andCry2Ab could only be achieved when both disabled toxins were added as anoverlay. When both tissues samples were combined at an LC50 value foreach sample, loss of inhibition was greater for the DT4 disabled toxinat a concentration of 2 mg/ml when compared to the loss of inhibitionfor the DT10 disabled toxin at the same concentration; suggesting TIC105plays a greater role in controlling Soybean looper than Cry2Ab.

Example 7

This example illustrates competition assay results between BCW003disabled toxin DT11 and homologous and heterologous toxin proteins.

The examples using Cry1Ab3 disabled toxin demonstrated that theactivities of BCW003 toxin (SEQ ID NO:34) and TIC105 toxin (SEQ ID NO:10) can be selectively abrogated depending on the insect species tested.The expanded set of Cry1A DP probes has enabled the evaluation ofheterologous competition among different insecticidal proteins, againusing SWC (southwest corn borer) as the test species due to itssensitivity to all of the insecticidal proteins included in thisexample. The disabled toxins corresponding to Cry1Ab3, TIC105, andBCW003 proteins each exhibited homologous competition with theirrespective native toxins from which the DT's were derived (see data inTable 11). Competition of DT's derived from these toxins (Cry1Ab3,TIC105, and BCW003) was not observed when tested with the more distantlyrelated Cry1Ca and Cry2Ab2 proteins. With respect to Cry1A heterologouscompetition, neither the TIC105_3 (SEQ ID NO:12) nor BCW003 disabledtoxin DT11 proteins exhibited competition with the TIC107 (Cry1Ac)native protein, suggesting that the unique domain 3 of TIC107 iscritical for the observed SWC inhibitory activity. It was also observedthat while Cry1Ab3_3 (SEQ ID NO:6) and TIC105_3 (SEQ ID NO:12)suppressed the activity of BCW003 (SEQ ID NO:34) toward SWC, DT11, theBCW003 disabled toxin variant, failed to suppress the activity of TIC105in the SWC feeding assay.

TABLE 11 Competition Against FFPP Amino Acid Toxin in SWCB SEQ ID NO:/Sequence Active on 1:1 Cry protein Modification SWCB 1:25 34/BCW003 — +NA 36/DT11 I109C, E129C − +/− + 12/TIC105 — + NA 12/DT11 I109C, E129C −− − 14/TIC107 — + NA 14/DT11 I109C, E129C − − − 18/Cry2Ab — + NA 18/DT11I109C, E129C − − − 38/Cry1Ca — + NA 38/DT11 I109C, E129C − − −

Example 8

This example illustrates competition assay results between Cry1Cadisabled toxin DT12 and homologous and heterologous toxin proteins.

Domain 1 sequences of Cry1Ca (SEQ ID NO:38) and Cry2Ab2 (SEQ ID NO:18)are very different from that of the Cry1Ab3 (SEQ ID NO:2) Domain 1(57.6% and 25.3% identity, respectively). Structural information aboutthese proteins was used to select a set of double cysteine mutations foreach such toxin so that the two thiols are positioned and orientedfavorably for EBI crosslinking. Taking into account structuralflexibility, residue pairs were selected with β-carbon atoms (Cβ) 7.3-12Å apart and pointing in the same direction. In cases where reactivity ofthe proposed amino acid substitutions might be sterically hindered bysurrounding bulkier residues, the potentially conflicting residues weremodified to smaller amino acids such as alanine or serine depending onthe expected hydrophobicity at those positions. 61 Cry1Ca and 44 Cry2Ab2designs were created and expressed in acrystalliferous Bacillusthuringiensis, and the variants that expressed well were tested ininsect diet bioassay. The Cry1Ca variants were tested on SWC and theCry2Ab2 variants were tested on CEW at an applied concentration of 1000ppm. Knowing in advance that the Cry1A and toxins related to Cry1Adisabled toxin candidates were shown to be inactive withoutcrosslinking, non-crosslinked Cry1Ca and Cry2Ab2 variants were testedfirst and the results identified several promising (i.e. inactive)protein variants for the follow-up homologous competition assay. Cry1Cadisabled toxin (DT12, SEQ ID NO:40) having the amino acid substitutions(N98C and D143C, and the Cry2Ab2 disabled toxin Cry2Ab26 (SEQ ID NO:30)having the amino acid substitutions G119C, N123A, L156C, and R160Asatisfied the criteria for use as disabled proteins for competitionstudies, exhibiting homologous competition in insect assays with Cry1Caand Cry2Ab2, respectively, at a high molar excess of the DT but not at a1:1 molar ratio. Comparative analysis between Cry1Ca and Cry2Ab2 usingtheir respective DT probes in the C. includens feeding assay showed thatthe respective DT for each protein was only able to compete out theactivity of the native protein from with the applicable DT was derived;i.e. Cry2Ab_6 was only able to compete with the Cry2Ab native protein,and DT12 was only able to compete with Cry1Ca.

Example 9

This example teaches in vivo receptor binding assessment via competitionassays between FAW-active insecticidal proteins and their respective DPvariants. TIC844 (SEQ ID NO:42), when provided in the diet to FAW larvaeat 690 ng/cm2 elicited a 98% insect stunting response, that wascalculated based on the observed insect size in reference to the size ofthe positive control (100% response) and negative control (0% response).DP assays were then implemented to comparatively assess the receptorpreferences of these native insecticidal proteins, and the proteins usedand the data collected is shown in Table 13. TIC844 provided at 690ng/cm2 when co-administered with DT13 (SEQ ID NO:44) exhibited (i) nocompetition at stoichiometric DP (1:1) to native insecticidal proteinratios, (ii) significant competition when DP was used 5-25 fold inexcess of the native insecticidal protein, and (iii) full competitionwhen the DP was used in 50-fold excess of the native toxin, where theinsect phenotype was completely rescued, and the insect size wasindistinguishable from the size of the negative control insects. WhenDT13 was co-administered separately with 5520 ng/cm2 TIC868 (SEQ IDNO:46), 20.7 ng/cm2 TIC842 (SEQ ID NO:50) and 2760 ng/cm2 Vip3A (SEQ IDNO: 54) (approximately an MIC95 dose), the insecticidal activity ofthese proteins was not inhibited, even in the presence of 138,000 ng/cm2DT13 competitor, representing a 25-, 6,600-, and 50-fold DP to thenative toxin challenge ratio, respectively. Similarly, homologouscompetition between native insecticidal proteins and their correspondingDIP variant was demonstrated for TIC868, TIC842, Vip3A, TIC105, andCry2Ab. Heterologous competition was also assessed between each nativeand DP pairs, and the insecticidal activity of TIC844, TIC868, TIC842,and Vip3A was not inhibited even in the presence of high concentrationDP competitor. These insecticidal proteins were evaluated against thedisabled version of two commercial insecticidal proteins, TIC105 (SEQ IDNO:10) and Cry2Ab (SEQ ID NO:18). Significant (P<0.05) competition wasnot observed with the exception of the comparison between 690 ng/cm2TIC844 and 138,000 ng/cm2 TIC1053 (SEQ ID NO: 12), which showed a mere15% reduction of insecticidal response under experimental conditionswhere TIC105_3 fully competed against its native counterpart.

TABLE 13 Competition SEQ ID NO:/ Against the Cry protein - correspondingAlone (A) or Amino Acid FFPP Toxin in Parent/DT Sequence FAWFAW/(Challenge Mixtures (M) Modification Activity Ratio) 42/TIC844 (A)S282V, Y316S, I368P + NA 46/TIC868 (A) — + NA 50/TIC842 (A) — + NA54/Vip3A(A) — + NA 10/TIC105 (A) — + NA 18/Cry2Ab (A) — + NA 44/DT13 (A)V108C, E128C, S282V, − NA Y316S, I368P 48/DT14 (A) A160N, N167D − NA52/DT15 (A) I108C, D128C − NA 56/DT16 (A) S175C, L177C − NA 12/DT4 (A)I109C, E129C − NA 30/DT10 (A) R129Q, R139Q, − NA G119C,N123A, L156C,R160A 42/TIC844 and NA + −/(Low)  44/DT13 (M) − +/(High) 42/TIC844 andNA + −/(Low)  48/DT14 (M) + −/(High) 42/TIC844 and NA + −/(Low)  52/DT15(M) + −/(High) 42/TIC844 and NA + −/(Low)  56/DT16 (M) + −/(High)42/TIC844 and NA + −/(Low)  12/DT4 (M) + −/(High) 42/TIC844 and NA +−/(Low)  30/DT10 (M) + −/(High) 46/TIC868 and NA + −/(Low)  44/DT13(M) + −/(High) 46/TIC868 and NA + −/(Low)  48/DT14 (M) − +/(High)46/TIC868 and NA + −/(Low)  52/DT15 (M) + −/(High) 46/TIC868 and NA +−/(Low)  56/DT16 (M) + −/(High) 46/TIC868 and NA + −/(Low)  12/DT4 (M) +−/(High) 46/TIC868 and NA + −/(Low)  30/DT10 (M) + −/(High) 50/TIC842and NA + −/(Low)  44/DT13 (M) + −/(High) 50/TIC842 and NA + −/(Low) 48/DT14 (M) + −/(High) 50/TIC842 and NA + −/(Low)  52/DT15 (M) −+/(High) 54/Vip3A and NA + −/(Low)  44/DT13 (M) + −/(High) 54/Vip3A andNA + −/(Low)  48/DT14 (M) + −/(High) 54/Vip3A and NA + −/(Low)  56/DT16(M) − +/(High)

Example 10

TABLE 14 Competition Data between Native Protein and Disabled ToxinCounterpart in Soybean Looper Larvae Homologous Competition Against theAmino Acid corresponding FFPP SEQ ID NO:/ Sequence SBL Toxin in SBL/ Cryprotein Modification Mortality (Challenge Ratio) 58/TIC1100 — + NA60/DT17 E99C, R144C − −/(Low) +/(High) 62/TIC867 — + NA 64/DT18 A160N,N167D − −/(Low) +/(High)

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of the foregoing illustrative embodiments, itwill be apparent to those of skill in the art that variations, changes,modifications, and alterations may be applied to the composition,methods, and in the steps or in the sequence of steps of the methodsdescribed herein, without departing from the true concept, spirit, andscope of the invention. More specifically, it will be apparent thatcertain agents that are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope, and concept of the invention as defined by theappended claims.

It should be apparent to those skilled in the art that these different,sequence variations can be combined to create variants which are alsowithin the scope of this invention.

All publications and published patent documents cited in thespecification are incorporated herein by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

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What is claimed is:
 1. A method for selecting two pesticidal agentscompatible for use together in a composition for controlling a targetpest, said method comprising: a) selecting a first and a second toxicagent, each agent being different from the other and each agent causingtoxic properties when provided individually in the diet of a targetpest; b) producing a DT from said first toxic agent that, upon ingestionby said target pest, blocks the toxic properties conferred by said firsttoxic agent but does not itself confer toxic properties; c) producing aplurality of different mixtures containing a fixed but pesticidallyeffective amount of a second toxic agent, and increasing amounts of saidDT; d) providing in the diet of said target pest, a pesticidallyeffective amount of said second toxic agent; e) providing a dose of eachmixture of step c) separately to each of at least three differentindividuals of said target pest; wherein observing toxic properties inany individual in step e) is determinative that said first and secondtoxic agents are compatible for use together to control said targetpest.
 2. A recombinant plant or seed expressing two or more pesticidalagents, wherein said agents are selected for use together as compatibleagents according to the method of claim
 1. 3. Selecting a first FFPP anda second FFPP to be combined together in a composition or for use on orin a plant to control insect infestation of said plant by a targetinsect pest species, wherein said combination of said FFPP's optionallyprovide: (a) for decreasing the likelihood of the development ofresistance by said target insect pest to any of the FFPP's in saidcomposition; (b) for aiding in or improving resistance managementpractices for controlling said target insect pest; or (c) for delayingthe onset of resistance to any of the FFPP's in said composition;Wherein (i) said two or more FFPP's are selected for use together by thesteps of claim 1; (ii) said DP, when used in a plurality of molar ratioswith a constant amount of said first FFPP, is effective in titering thetoxic effect of the first FFPP upon said target pest; and (iii) said DP,when used in a plurality of molar ratios with a constant amount of saidsecond FFPP, does not titer the toxic effect of the second FFPP uponsaid target pest; and Wherein said first and said second FFPP arecompatible for use together in said composition on or in said plant. 4.A method of assessing the mode of action of a first FFPP forcompatibility with a second FFPP to be used in a common pesticidalcomposition, said method comprising the steps of: (a) preparing a DPfrom a first FFPP that is toxic to a target pest; (b) confirming thatsaid DP when used alone in a bioassay with said target pest hasdiminished toxicity against the target pest when compared to thetoxicity of the first FFPP; (c) comparing said DP to a second FFPPdifferent from said first FFPP alone and in a plurality of molar ratiosin which the DP is present in a greater concentration than said secondFFPP in the diet of the target pest; wherein the inability to titer thetoxic properties of said second FFPP with said DP is determinative ofthe binding of said first FFPP and said second FFPP to differentreceptors in said target pest.
 5. The method of any of claim 3 or 4,wherein a composition comprising said first FFPP and said second FFPP iseffective in controlling an insect pest infestation wherein said insectsare selected from the group consisting of Arachnida, Coleoptera,Ctenocephalides, Diptera, Hemiptera, Heteroptera, Homoptera,Hymenoptera, Lepidoptera and Thysanoptera insects.
 6. The method ofclaim 1, wherein a composition comprising said first toxic agent andsaid second toxic agent is effective in controlling an insect pestinfestation wherein said insects are selected from the group consistingof Arachnida, Coleoptera, Ctenocephalides, Diptera, Hemiptera,Heteroptera, Homoptera, Hymenoptera, Lepidoptera and Thysanopterainsects.
 7. A plant or a seed from a plant comprising a firstrecombinant nucleic acid molecule comprising a first heterologouspromoter operably linked to a first polynucleotide segment encoding afirst FFPP and a second recombinant nucleic acid molecule comprising asecond heterologous promoter operably linked to a second polynucleotidesegment encoding a second FFPP different from said first FFPP, whereinsaid first FFPP and said second FFPP are selected for use together fromthe steps as set forth in any of claim 3 or 4, wherein, optionally: a)said plant or said seed are produced from the breeding together by thehand of man of two different plants of the same or substantially similarspecies, a first plant comprising said first recombinant nucleic acidmolecule expressing said first FFPP and a second plant comprising saidsecond recombinant nucleic acid molecule expressing said second FFPP; b)said plant or seed are produced from the regeneration of a plant fromthe transformation of a first plant cell by said first recombinantnucleic acid molecule and the transformation of a second plant cell bysaid second recombinant nucleic acid molecule; c) said plant or seed areproduced from the transformation of a single plant cell by said firstand said second recombinant nucleic acid molecule; and d) said plant orseed are grown from a plant or seed of any of a), b), or c), whereinsaid plant or seed comprise said first and said second recombinantnucleic acid molecule.
 8. A composition comprising a first polypeptideand a second polypeptide that is different from the first polypeptide,wherein said first and second polypeptides are each toxic to a targetpest and do not bind to the same target receptor in said pest, andwherein said first and second polypeptide are selected for use togetherby the steps of: a) producing a DT from said first polypeptide that,upon ingestion by said target pest, blocks the toxic propertiesconferred by said first polypeptide but does not itself confer toxicproperties; b) producing a plurality of different mixtures containing afixed but pesticidally effective amount of said second polypeptide, andincreasing amounts of said DT; c) providing in the diet of said targetpest, a pesticidally effective amount of said second polypeptide; d)providing a dose of each mixture of step c) separately to each of atleast three different individuals of said target pest; wherein observingtoxic properties in any individual in step e) is determinative that saidfirst and second polypeptides are compatible for use together to controlsaid target pest.